Expression and function of B7-1 and B7-2 in hapten-induced contact sensitivity

We analyzed the expression and function of the co-stimulatory molecules B7-1 (CD80) and B7-2 (CD86) during contact sensitivity reactions induced by the hapten 2,4-dinitrofluorobenzene (DNFB). In the normal skin, only a few epidermal Langerhans cells or dermal dendritic cells express B7-2. In contrast, following challenge with DNFB, expression of B7-2 is up-regulated in both epidermis and dermis. Importantly, B7-1 is induced later and at lower levels compared to B7-2. Intravenous injections of anti-B7-2 mAb, but not anti-B7-1 mAb partially inhibit the hapten-induced contact sensitivity reaction. Experiments in which mice are injected differentially with anti-B7-2 mAb, either before the afferent or before the efferent phase of the contact sensitivity response, suggest that B7-2 is important for successful antigen priming.     

Mitogenic stimulation of T cells reveals differing contributions for B7-1 (CD80) and B7-2 (CD86) costimulation.

The requirement of accessory cells for concanavalin A (Con A) activation of T cells suggests delivery of a separate costimulatory signal. However, the costimulatory pathways involved have not been identified. These studies assess the role of CD28-B7-mediated costimulation during T-cell activation by Con A. The B7-1/B7-2 binding protein CTLA4-Ig inhibited the proliferative response of primary lymph node cells to either Con A or soluble anti-CD3 mAb. This suppression was dose dependent and could be reversed by CD28 cross-linking. CTLA4-Ig also completely suppressed induction of interleukin-2 (IL-2) mRNA by Con A. CTLA4-Ig-mediated suppression was not due to blockade of the Con A 'receptor(s)' or of the primary activation signal (as measured by the intracellular calcium response). Although both B7-1 and B7-2 were up-regulated following Con A activation, each played a different role in proliferation and cytokine production. Individually, anti-B7-2 Fab partially inhibited the Con A response whereas anti-B7-1 Fab had no effect. However, the combination of anti-B7-1 and anti-B7-2 Fab completely suppressed proliferation and IL-2 production. Therefore, while a part of the Con A response requires B7-2, the remainder of the response can utilize either B7-1 or B7-2. Together, these results demonstrate that Con A activation of T cells requires the delivery of a separate costimulatory signal that is mediated almost entirely by the B7 receptors.     

Prevention and amelioration of collagen-induced arthritis by blockade of the CD28 co-stimulatory pathway: requirement for both B7-1 and B7-2

Collagen type II-induced arthritis (CIA) is an experimental model of arthritis that has been successfully used to dissect the pathogenesis of human rheumatoid arthritis and to identify potential therapeutic targets. We have used this model to evaluate the role of T cell co-stimulation in both disease development and progression. T cell co-stimulation is provided by ligation of CD28 with either B7-1 or B7-2 present on antigen-presenting cells and can be prevented by a soluble form of CTLA-4 (CTLA-4Ig) which binds with high affinity to both B7-1 and B7-2. We found that administration of CTLA-4Ig at the time of immunization prevented the development of CIA and was associated with lack of lymphocyte expansion within the draining lymph node and failure to produce anti-collagen IgG1 or IgG2a antibodies. To determine which CD28 ligand plays a more dominant role in CIA, we treated mice with monoclonal antibodies (mAb) against either B7-1 or B7-2. Neither anti-B7-1 nor anti-B7-2 had any effect on the course of CIA when given alone, but resulted in reduced incidence and clinical scores when given together. Interestingly, when treatment was delayed until after the onset of clinical disease, both CTLA-4Ig or anti-B7-1 plus anti-B7-2 mAb still ameliorated disease. Effective treatment was associated with a reduction in interferon-γ production by lymph node cells following stimulation in vitro, suggesting that Th1 responses were diminished. This study points to a critical role of CD28 co-stimulation in the development and perpetuation of CIA in DBA/1 mice. Interestingly, it demonstrates an active role for T cells in the later stages of this disease and implicates both B7-1 and B7-2-mediated co-stimulation in the pathogenesis of CIA.     

Both CD28 ligands CD80 (B7-1) and CD86 (B7-2) activate phosphatidylinositol 3-kinase, and wortmannin reveals heterogeneity in the regulation of T cell IL-2 secretion

In this report, the co-stimulatory signals provided by CD80(B7-1) or CD86 (B7-2) were compared to CD28 ligation by mAb.We demonstrate that while both anti-CD3 and anti-CD28 antibodiesinduced activation of phospholnositide (PI) 3-kinase, the kineticsof activation differed. Anti-CD28 produced a sustained activationof PI 3-kinase while anti-CD3 induced activation was transient.Both B7-1 and B7-2 could induce prolonged activation of PI 3-kinase.The co-stimulatory effects of B7-1 and B7-2 were dependent onCD28 cross-linking, based on complete inhibition of PI 3-kinaseactivation by CD28 antibody Fab fragments. While Jurkat T cellsco-stimulated with anti-CD3 and B7-1 or B7-2 secreted high levelsof IL-2, there were distinct effects of anti-CD28 mAb and B7-1or B7-2 on IL-2 secretion in conjunction with protein kinaseC activation. To assess functional effects of CD28 ligation,pharmacologic inhibitors of PI 3-kinase were evaluated. In Jurkatcells, efficient inhibition of PI 3-kinase activation afterB7-2 stimulation was achieved using wortmannin; however, weobserved a surprising increase in IL-2 secretion after B7 oranti-CD28 stimulation. The effect of wortmannin was concentrationdependent. Moreover, the effect was specific for receptor-mediatedactivation as wortmannin did not enhance phorbol ester pluslonomycin-induced IL-2 secretion. Another inhibitor of PI 3-kinase,LY294002, also resulted in augmentation of anti-CD28-inducedIL-2 secretion by Jurkat cells. The effects of wortmannin onIL-2 secretion were also examined in primary T cells. In markedcontrast, wortmannin resulted in a potent inhibition of anti-CD3plus B7-1 or anti-CD28-induced IL-2 secretion while phorbolester plus lonomycin-induced IL-2 secretion was wortmannin resistant.Together these observations demonstrate that signal transductionby both B7-1 and B7-2 involves PI 3-kinase, and that PI 3-kinaseor other wortmannin-sensitive targets are important for IL-2secretion. Finally, treatment of Jurkat cells with PI 3-kinaseinhibitors alone was sufficient to induce low levels of IL-2secretion. This is consistent with the notion that a wortmannin-sensitivetarget such as PI 3-kinase may down-regulate IL-2 secretionin Jurkat cells.     

CD28 ligands CD80 (B7-1) and CD86 (B7-2) induce long-term autocrine growth of CD4+ T cells and induce similar patterns of cytokine secretion in vitro

The interaction of CD28 and its ligands is critical for antigen-inducedT cell activation. Recent studies have demonstrated the existenceof at least two members of the B7 receptor family. In this report,the co-stimulatory signals provided by CD80 (B7-1) or CD86 (B7-2)were compared to CD28 ligation by mAb. We demonstrate that thekinetics of induction of T cell proliferation after anti-CD3stimulation was similar regardless of the form of co-stimulation.Similarly, B7-1 and B7-2 could both maintain long-term expansionof CD4 cells. The co-stimulatory effects of both B7-1 and B7-2were dependent on CD28 cross-linking, based on complete inhibitionof proliferation by CD28 antibody Fab fragments. Co-stimulationwith B7-1 and B7-2 induced high levels of cytokine secretionby resting T cells, and the effects of B7-1 and B7-2 could notbe distinguished. This conclusion is based on analysis of theinitial activation of CD28⁺ T cells. as well as T cell subpopulationsconsisting of CD4⁺ and CD8⁺ T cells. Both B7-1 and B7-2 couldelicit IL-4 secretion from CD4⁺ T cells while anti-CD28 antibodyinduced substantially less IL-4 secretion. Furthermore, bothB7-1 and B7-2 could stimulate high levels of IFN- and IL-4 fromCD4⁺CD45RO⁺ cells, while neither B7 receptor could co-stimulateIFN- and IL-4 secretion from CD4⁺CD45RA⁺ T cells. B7-1 and B7-2could, however, co-stimulate CD4⁺CD45RA⁺ T cells to secreteIL-2. By contrast, when previously activated T cells were tested,re-stimulation of CD4⁺ T cell blasts with B7-1 or B7-2 resultedin higher secretion of IL-4 and IL-5 than anti-CD28, while re-stimulationwith anti-CD28 antibody maintained a higher level of secretionof IL-2 and IFN- than B7-1 or B7-2. These observations may haveimportant implications because they suggest that the mannerof CD28 ligation can be a critical determinant in the developmentof cytokine secretion that corresponds to T_h1- and T_h2-likepatterns of differentiation. Together these observations suggestthat there are no Intrinsic differences between B7-1 and B7-2in their ability to co-stimulate the populations of cells thatwe have tested.     

Cryptococcus neoformans differently regulates B7-1 (CD80) and B7-2 (CD86) expression on human monocytes

To induce a specific response in primary resting T cells, two signals must be provided by antigen-presenting cells (APC). The first antigen-specific signal is mediated by formation of the T cell receptor major histocompatibility complex molecule ternary complexes. The second signal is delivered by interaction of either B7-1 or B7-2 expressed by APC with CD28 or CTLA-4 on T cells. In this study, we examined the modulation of B7-1 and B7-2 molecules on human monocytes exposed to encapsulated or acapsular Cryptococcus neoformans or Candida albicans. In our experimental system, C. albicans or acapsular C. neoformans are able to induce B7-1 expression while the encapsulated yeast is a poor stimulator. A modest increase of B7-2 expression was also observed after monocyte treatment with acapsular C. neoformans or C. albicans, while the encapsulated yeast was ineffective in inducing B7-2 molecules. Kinetic analysis showed the maximum expression of B7-1 after 24 to 48 h. Addition of the opsonic IgG1 mAb 2H1 to monocytes and C. neoformans significantly increased B7-1, but not B7-2, expression. The contribution of B7-1 and B7-2 co-stimulatory (CS) molecules to cryptococcal-specific T cell activation was analyzed and a substantial inhibition of T cell proliferation was observed. In this study we provide the first demonstration of fungal interference in the regulation of CS molecules. Our results suggest a potential mechanism for poor inflammatory responses observed in C. neoformans infections.     

Human B7-1 is more efficient than B7-2 in providing co-stimulation for alloantigen-specific T cells

Besides a signal via the T cell receptor/CD3 complex, an additional co-stimulatory signal is required for optimal T cell activation. This signal can be delivered by interaction of either B7-1 or B7-2 expressed by antigen-presenting cells with CD28 on the T cells. Comparison of the function of B7-1 and B7-2 in different experimental animal systems generated conflicting data on the roles for the co-stimulatory molecules. We therefore investigated whether there are differences between B7-1 and B7-2-mediated co-stimulation in an alloantigen-specific primary T cell response induced by B7-transfected human cell lines of epithelial origin. Both transfected keratinocyte cell lines efficiently induce T cell proliferation and the ratios of stimulator versus responder cells are similar. The kinetics of proliferation and interleukin (IL)-2, IL-4 and interferon-γ production are also comparable between both transfectant lines. However, despite equal B7 expression levels, it is consistently found that the magnitude of the B7-1-induced T cell proliferation was higher than that of B7-2. Comparison of precursor frequencies of helper T lymphocytes responsive with either B7-1 or B7-2 revealed that the frequency of B7-1-responsive T cells was higher than that of B7-2, and that the frequency of cells activated by a combination of B7-1 and B7-2 did not differ significantly from that of B7-1 alone. We therefore conclude that the B7-2-responsive T cells are part of the B7-1-responsive population, and that B7-1 on keratinocytes is more efficient in providing co-stimulation for alloantigen-specific T cells.     

Differential requirements for co-stimulatory signals from B7 family members by resting versus recently activated memory T cells towards soluble recall antigens

The interaction between CD28 on T cells with CD80 (B7-1) andCD86 (B7-2) on APCs is considered to be of critical importancefor primary T cell activation both in vivo and in vitro. Therelative importance of this co-stimulatory signal in memoryT cell activation is, however, less clear, and was thereforestudied by in vitro experiments on T cell responses to solublerecall antigens using peripheral blood mononuclear cells orT cell clones. Our data demonstrate that B7-2 represents themajor co-stimulatory signal for the activation of resting peripheralblood memory T cells with recall antigens, as evidenced by theeffects of anti-B7-1 and anti-B7-2 on T cell proliferation aswell as on IL-2 and INF-y production. Since CTLA-4-lg and anti-CD28Fab fragments had similar inhibitory effects to the combinationof anti-B7-1 plus anti-B7-2, the involvement of a third co-stimulatoryCD28/CTLA-4 ligand is unlikely. Despite the strong effects ofB7-blocking agents, a variable fraction of the memory T cellswas resistant to inhibition. Moreover, T cell clones or in vitropreactivated T cells could efficiently be restimulated by solubleantigens on autologous APCs in the absence of B7-1 or B7-2 co-stimulation.These data show that most memory T cells that are freshly isolatedfrom the blood are still dependent on CD28 triggering for theiractivation. However, recently activated T cells can apparentlybypass the requirement for B7 and use other co-stimulatory signalsfor reactivation, a finding with important implications forthe development of immunosuppressive strategies.     

Anti-B7-1/B7-2 antibody elicits innate-effector responses in macrophages through NF-kappaB-dependent pathway

Blocking T cell co-stimulatory signals by anti-B7-1/B7-2 mAb is an attractive approach to treat autoimmune diseases. However, anti-B7-1/B7-2 mAb treatment is known to exacerbate autoimmune diseases through mechanisms not fully understood. Tumor necrosis factor alpha (TNF-alpha) and reactive oxygen species (ROS) also play important roles in determining the clinical outcome of autoimmune diseases. In this study, we demonstrate that the anti-B7-1 and the anti-B7-2 mAbs activate macrophages for higher induction of TNF-alpha and other effector responses such as bacterial cytotoxicity and production of ROS. Nuclear factor-kappaB (NF-kappaB) was found to be increased with anti-B7-1/B7-2 mAb treatment. Inhibition of NF-kappaB activity by over-expression of phosphorylation-defective I-kappaB alpha in anti-B7-1/B7-2 mAb-treated macrophages decreased TNF-alpha production. These data indicate that anti-B7-1 and anti-B7-2 mAbs can trigger innate-effector responses in macrophages by activating NF-kappaB-signaling pathway. Our results suggest that the B7 molecules are not only essential for induction of adaptive immune responses but also play roles in activation of innate immune responses.     

Ectopic Expression of B7-1 (CD80) on T Lymphocytes in AutoimmunelprandgldMice

Defective Fas-mediated apoptosis in mice, caused by the gld mutation in the fas ligand gene, results in the development of lupus-like autoantibodies and severe lymphoproliferation. We previously demonstrated ectopic expression of the costimulatory molecule B7-1 (CD80) on T lymphocytes in B6/gld mice. This report extends these observations by demonstrating similar results in B6/lpr mice, which possess a mutation in the gene encoding Fas. Additionally, we demonstrate that this phenomenon is age-dependent and occurs on multiple subsets of B6/gld T lymphocytes. B7-1 upregulation is observed on T cells from both conventionally housed and specific-pathogen-free B6/gld mice, suggesting that this is not a consequence of infection by pathogen. T cells from lpr and gld mice show increased binding of CTLA4-Ig fusion protein, suggesting that the upregulated B7-1 is functional. CD28, a receptor for B7-1 which activates T cells, is upregulated in B6/lpr and B6/gld mice, while CTLA4, a negative regulator of T cells which binds B7-1, is not. Our results suggest that ectopic expression of B7-1 on T cells of lpr and gld mice may be playing a role in exacerbation of lymphoproliferation and/or autoimmunity.     

Potential role of B7-1 and CD28 molecules in immunosuppression in leprosy

In order to understand the mechanism of unresponsiveness towards Mycobacterium leprae antigens in leprosy, we evaluated the role of M. leprae sonicate antigens in regulating the expression of the costimulatory molecules B7-1, CD28, intercellular adhesion molecule-1 (ICAM-1), LFA-1α, LFA-1β and Mac-1 on the lymphocytes of both leprosy patients and healthy subjects. It was observed that the expression of B7-1 and CD28 was significantly decreased but the levels of ICAM-1 and LFA-1α were increased in patients with untreated borderline leprosy (BL)/lepromatous leprosy (LL) disease. No remarkable change was noticed in the case of borderline tuberculoid (BT) leprosy or treated BL/LL patients. Further, a striking finding was that lymphocytes from healthy subjects cultured with a particularly high dose of M. leprae sonicate antigens down-regulated the expression of B7-1 and CD28 molecules, but up-regulated the display of ICAM-1 and LFA-1α. Furthermore, proliferation induced by M. leprae sonicate was inhibited only by anti-B7-1 antibody. Mycobacterium leprae antigen-induced suppression of the proliferation of lymphocytes of healthy volunteers and LL patients was reversed by culturing the lymphocytes with purified protein derivative (PPD). It may be concluded from the findings in this study that down regulation of B7-1 and CD28 in BL/LL leprosy patients may be responsible for a defective T cell signalling by the B7-1/CD28 pathway caused by M. leprae antigens. This may lead to clonal inactivation of M. leprae-reactive T cells, consequently the bacilli grow without restriction in macrophages.     

Localization in situ of the co-stimulatory molecules B7.1, B7.2, CD40 and their ligands in normal human lymphoid tissue

Functional interactions between B and T lymphocytes are known to depend on the expression of co-stimulatory molecules B7.1/CD80, B7.2/CD86 and their counter-receptors CD28 and CTLA4, as well as CD40 and its ligand CD40L. To study the role of these molecules in situ, an immunohistochemical analysis was carried out on normal human lymphoid tissue. In the germinal centers (GC), B7.1 and B7.2 were differentially expressed. In the dark zone, centroblasts were predominantly B7.1⁺, while centrocytes in the light zone were B7-2⁺, resulting in reversed gradients of both markers in GC. Follicle mantle cells were negative for B7.1 and B7.2. Macrophages and interdigitating dendritic cells (IDC) in T cell zones both expressed B7.1 and B7.2. Moreover, clusters of B7.2⁺ T cells were demonstrated in interfollicular areas. Intrafollicular CD4⁺ T cells in GC, predominantly in the apical light zone, expressed CD28 and CTLA4, as did the majority of interfollicular T cells. CTLA4 showed a striking excentric cytoplasmic staining, which was also seen on T cells activated in vitro. CD40 was expressed on all B cells and more strongly on macrophages and IDC. Moreover, small clusters of T cells in a rim outside the GC showed CD40 expression. CD40L was expressed both on intrafollicular CD4⁺ T cells as well as on T cells in T cell zones. The differential distribution of co-stimulatory molecules in different compartments of normal human lymphoid tissue in situ indicates that these interactions play a distinctive role in different stages of B cell differentiation and in the immune response.     

B7-1-dependent co-stimulation results in qualitatively and quantitatively different responses by CD4+ and CD8+ T cells

To characterize better the co-stimulatory activity of native B7-1 in the absence of other receptor/ligand interactions that might contribute to the response, B7-1 was purified by monoclonal antibody (mAb) affinity chromatography. Immobilization of purified B7-1 with anti-T cell receptor (TCR) mAb on cell-sized latex microspheres provided an effective stimulus for activation of both CD4⁺ and CD8⁺ T cells as measured by proliferation, development of effector function, and changes in motility and adhesion. The CD4⁺ T cell response was prolonged and resulted in efficient interleukin-2 production and clonal expansion. In contrast, CD8⁺ responses were transient. Proliferation and clonal expansion peaked on days 3 and 4, coincident with maximal expression of lytic effector function, and the cells then died. These results demonstrate that B7-1 mediated co-stimulation is sufficient for the induction of effector function in both helper and cytotoxic T cell precursors, but suggest that B7-1 co-stimulation is not sufficient to sustain helper-independent CD8⁺ CTL responses. When the dose responses of CD4⁺ and CD8⁺ T cells to B7-1 were compared, CD8⁺ T cells were found to require higher densities of B7-1 to attain an equivalent level of activation, suggesting that the level of expression of B7-1 by APC may influence the development of helper or CTL responses. Finally, in contrast to results obtained by others with B7-1 transfectants, purified B7-1 did not provide co-stimulation when presented on a surface separate from the TCR stimulus.     

Primary porcine endothelial cells express membrane-bound B7-2 (CD86) and a soluble factor that co-stimulate cyclosporin A-resistant and CD28-dependent human T cell proliferation

Increasing evidence suggests that endothelial cells can directlyactivate syngeneic, allogeneic and xenogeneic T cells. In thisstudy we demonstrate that unstimulated, paraformaldehyde-fixedprimary porcine aortic endothelial cells (PAEC) and microvascularendothelial cells (PMVEC) can provide co-stimulation for humanT cell IL-2 secretion and proliferation. EC-mediated co-stimulationhas both cyclosporin A (CsA)-sensitive and CsA-resistant components.The CsA-resistant component is completely suppressed eitherby blocking with anti-CD28 F(ab) fragments or CTLA-4-lg. Northernblot analysis of unstimulated PAEC and PMVEC with porcine-specificprobes reveals constitutive expression of B7-2 mRNA while B7-1message was not detected. hCTLA-4-lg and anti-B7-2 mAb immunoprecipitatesa single 79 kDa PMVEC surface protein. Surprisingly, PMVEC conditionedmedia also has soluble co-stimulatory activity that is blockedby anti-CD28 F(ab) fragments or anti-B7-2 mAb. These findingsdemonstrate that primary unstimulated porcine EC can co-stimulateCsA-resistant human T cell proliferation through binding ofmembrane bound, constitutively expressed EC B7-2 (CD86) to humanT cell CD28, providing one of the first demonstrations of functionalB7-2 on cells outside the immune system. In addition, PMVECsecrete or shed a soluble factor that mediates CD28-dependenthuman T cell proliferation, demonstrating the existence of solublemediators of CD28 activation.     

Studies on the interdependence of gp39 and B7 expression and function during antigen-specific immune responses

Interactions between T and B cells are dynamic and regulated by interacting receptor: co-receptors. Interactions between CD40 and its ligand, gp39, and the CD28/CTLA-4 and B7 family members play a decisive role in regulating the progression of cognate interactions. The interdependence of gp39-CD40 and CD28/CTLA-B7 expression and function was studied in vitro during an antigeninduced immune response using T cells from mice expressing a transgenic T cell receptor (TCR). gp39 was induced on pigeon cytochrome c (PCC)-transgenic T cells in the presence of antigen and antigen-presenting cells. The antigen-induced expression of gp39 on transgenic T cells was inhibited by antibodies to class II major histocompatibility complex, CD4 and LFA-1, but not by CTLA-4 Ig, anti-B7-1 or anti-B7-2. These data established that the antigen-induced expression of gp39 was not dependent on co-stimulation via CD28/CTLA-4. The addition of PCC also resulted in the modest expression of B7-1 and a more robust expression of B7-2 on the cognate B cells. The addition of anti-gp39 blocked the up-regulated expression of B7-1 and partially blocked the up-regulated expression of B7-2. The addition of anti-gp39 and anti-interleukin-4 inhibited antigen-induced expression of B7-2 on B cells to near background levels. Studies on the up-regulation of B7-1 and B7-2 on resting B cells showed that soluble gp39 up-regulated B7-1 and B7-2 expression on B cells. In addition, interleukin-4 and interferon-γ up-regulated B7-2 expression on B cells. Taken together, these data demonstrate that the antigen-induced expression of gp39 is dependent on TCR-derived signals, yet independent of CD28/CTLA-4 co-stimulatory signals. Cognate interactions also resulted in the modest enhancement of B7-1 expression and a more profound expression of B7-2 which were completely or partially dependent on gp39-CD40 interactions.     

Costimulatory molecules in Wegener's granulomatosis (WG): lack of expression of CD28 and preferential up-regulation of its ligands B7-1 (CD80) and B7-2 (CD86) on T cells

T cells are most likely to play an important role in the pathogenesis of WG, and recently a predominant Th1 pattern of immune response has been demonstrated in granulomatous inflammation. Since the expression of costimulatory molecules has a significant impact on the cytokine profile and proliferation response of T cells, the goal of this study was to characterize the expression of costimulatory molecules (CD28, CTLA-4 (CD152), B7-1 (CD80), B7-2 (CD86)) on T cells, monocytes and B cells in WG, and to correlate the findings with clinical parameters such as disease activity, extent and therapy. WG patients (n = 24) and healthy controls (HC; n = 17) were examined for the expression of costimulatory molecules by fluorescence-activated cell sorter analysis, both in whole peripheral blood and after in vitro activation of T cells and antigen-presenting cells. Results were correlated with clinical data. The expression of CD28 on CD4⁺ and CD8⁺ cells was significantly lower in WG than in HC (CD28⁺ 81.4% in WG versus 97.9% of CD4⁺ cells (P < 0.0001); CD28⁺ 44.6% in WG versus 68.5% of CD8⁺ cells (P < 0.00001)), both in peripheral blood and after in vitro activation. A lower percentage of monocytes was B7-2⁺ in WG than in HC in peripheral blood, whereas no significant differences in the expression of B7-1 and B7-2 were observed after in vitro stimulation of monocytes and B cells. After in vitro activation a significantly higher percentage of B7-1⁺ and B7-2⁺ T cells was seen in WG. There was no significant difference in the CTLA-4 expression pattern between WG and HC. The percentage of CD28⁺ lymphocytes correlated negatively with the Disease Extent Index cumulated over the course of disease (r = ?0.46, P = 0.03), indicating a more severe manifestation in patients with lower CD28 expression. Correlations with other clinical parameters such as activity or therapy were not seen. WG patients show a lack of CD28 expression on T cells and an unusual up-regulation of its ligands B7-1 and B7-2 on T cells after in vitro activation as well as a lower expression of B7-2 on freshly isolated monocytes compared with HC. These features might promote the Th1 cytokine pattern and thereby contribute to persistently high levels of immune activation in WG.     

B7-CD28 interaction is a late acting co-stimulatory signal for human T cell responses

The interaction of CD28 with one of the B7 molecules (CD80 and CD86) on professional antigen-presenting cells (APC) is generally considered as the most important co-stimulatory signal for T cell activation. APC in a resting condition express either no or only low levels of B7 molecules. These are up-regulated as a result of interactions with activated T cells, thus suggesting that B7-CD28 interaction is not required at initiation of T cell activation. To study this issue, we blocked B7-CD28 interaction at various time points after in vitro stimulation of peripheral blood T cells with allogeneic monocytes. Epstein-Barr virus-transformed B cells or soluble antigens. We observed that T cell proliferation and IL-2 production were inhibited by B7- blocking agents (CTLA-4-Ig or anti-B7 mAb) almost to the same degree when added either at initiation of culture or 24 h later. B7-blocking agents still resulted in significant inhibition of allogeneic T cell activation when added after 48 h. Furthermore, when CTLA-4-Ig was added at the start of an allogeneic T cell stimulation, addition of anti-CD28 mAb after 24 h of culture nearly fully restored T cell proliferation to control levels. Finally, we demonstrate that delayed addition of B7- blocking agents together with cyclosporin A 1 day after the onset of culture of T cells with allogeneic B cells is highly efficient to induce energy as evaluated by lack of proliferation, cytotoxic T lymphocyte reactivity and IFN-gamma or IL-5 production upon alloantigen rechallenge. Taken together, our data can explain why B7 expression on APC is not required at the time of initial APC-T cell contact, and suggest that the effect of the CD28 signal indeed consists in prolonging IL-2 production and amplifying T cell responses, rather than in providing a critical co-stimulatory signal at the time of initial TCR triggering.      

Differential effects of B7-1 and B7-2 on the costimulation of mouse nonspecific cytotoxic T lymphocyte development in response to anti-CD3 antibody

Despite extensive study, the relative contribution of B7-1 and B7-2 molecules to the costimulation of cytotoxic T lymphocyte (CTL) activation remains controversial. We used blocking mAbs to B7-1 and B7-2 molecules to determine the role of these B7 family members in the in vitro induction of mouse nonspecific CTL in response to soluble anti-CD3 mAb. Optimal induction of anti-CD3-activated killer-T (AK-T) cells was found to require interactions with B7-2 on residual accessory cells in nylon wool-nonadherent spleen cell preparations during the first 12 h of culture in the presence of anti-CD3 mAb. Because B7-1 is not expressed at high enough levels on residual accessory cells in primary T cell cultures to be an effective ligand for CD28, we used LPS-stimulated B cells, which express substantial B7-1, in addition to B7-2, to determine the contribution of B7-1 to AK-T cell development. Compared with B7-2, the contribution of B7-1 to the costimulation of AK-T cells in this system was modest because anti-B7-1 mAb had only a minimal inhibitory effect on the generation of cytotoxicity, whereas anti-B7-2 mAb strongly inhibited AK-T cell development. Anti-CD3-induced cytotoxicity of T cells from CD4 knockout mice and CD4-depleted nylon wool-nonadherent spleen cells from wild-type mice was inhibited by anti-B7-2 mAb, implying that B7-2 is able to bind directly to CD28 on CD8+ T cells and costimulate their activation. B7-1 blockade, on the other hand, did not affect the costimulation of CD8+ T cells. Blockade of B7-2/ CD28 interactions with anti-B7-2 mAb strongly inhibited granzyme B, but not perforin or Fas ligand gene expression, suggesting an explanation for the inhibitory effect of anti-B7-2 mAb on AK-T cell development. These data indicate that B7-2 is superior to B7-1 as a costimulator of mouse AK-T cell induction.