Activated keratinocytes in the epidermis of hypertrophic scars

CD40-CD40-ligand (CD154) interactions play a critical role in immune activation. Using replication defective adenovirus encoding mouse CD154 (Ad-CD154), we modified human chronic lymphocytic leukemia B cells to express a functional ligand for CD40. This not only induces expression of immune accessory molecules on the infected cell, but also allows it to trans-activate noninfected bystander leukemia B cells. Also, factors that impair the antigen-presenting capacity of leukemia B cells are downmodulated. Ad-CD154- infected leukemia cells are highly effective stimulators in mixed lymphocyte reactions and can induce generation of cytotoxic T lymphocytes specific for autologous nonmodified leukemia cells. As such, Ad-CD154 can induce a host antileukemia response that may have therapeutic potential.     

Antitumor effect of CD40 ligand: elicitation of local and systemic antitumor responses by IL-12 and B7

The interaction between CD40 ligand (CD40L, CD154) and its receptor CD40 has been implicated in the establishment of cell-mediated immunity as well as humoral immune responses. To examine the role of CD40L in eliciting antitumor immunity, we introduced murine CD40L gene into P815 mastocytoma (CD40L-P815). CD40L-P815 cells underwent prompt rejection when inoculated s.c. into syngenic DBA/2 mice or athymic BALB/c nu/nu mice, which was mediated by NK cells and dependent on endogenous IL-12. The primary rejection of CD40L-P815 cells in DBA/2 mice elicited CD8+ T cell-mediated protective and systemic immunity against parental tumor cells, which was induced by CD4+ T cells and endogenous B7. These results indicated a potent antitumor effect of CD40L that is mediated by potentiation of host Ag-presenting cell functions, and introduction of CD40L will be useful as a new strategy of immuno-gene therapy against tumors.     

IL-15 promotes IL-12 production by human monocytes via T cell-dependent contact and may contribute to IL-12-mediated IFN-gamma secretion by CD4+ T cells in the absence of TCR ligation

At inflammatory sites, the number of activated bystander T cells exceeds that of Ag-activated T cells. We investigated whether IL-15, a monocyte-derived cytokine that shares several biologic activities with IL-2, may contribute to bystander T cell activation in the absence of IL-2 and triggering Ag. The addition of IL-15 to cocultures of monocytes and T cells stimulates CD4+ but not CD8+ T cells to produce IFN-gamma. IFN-gamma production requires endogenous IL-12, the production of which in turn is dependent upon CD40/CD154 interactions between CD4+ T cells and monocytes. Indeed, non-TCR-activated CD4+ but not CD8+ T cells express significant levels of CD154. IL-15 may enhance IFN-gamma in this system by up-regulating CD40 expression on monocytes and IL-12Rbeta1 expression on CD4+ T cells. Conversely, using neutralizing anti-IL-15 mAb, we show that the ability of IL-12 to augment IFN-gamma secretion is partly mediated by endogenous IL-15. Finally, in the absence of monocytes, a synergistic effect between exogenous IL-12 and IL-15 is necessary to induce IFN-gamma production by purified CD4+ T cells, while IL-15 alone induces T cell proliferation. It is proposed that this codependence between IL-12 and IL-15 for the activation of inflammatory T cells may be involved in chronic inflammatory disorders that are dominated by a Th1 response. In such a response, a self-perpetuating cycle of inflammation is set forth, because IL-15-stimulated CD4+ T cells may activate monocytes to release IL-12 that synergizes with IL-15 to induce IL-12 response and IFN-gamma production.     

Blockade of CD40-CD40 ligand pathway induces tolerance in murine contact hypersensitivity

Interactions between CD40 on antigen-presenting cells and its ligand (CD40L) on T cells has been implicated in T cell-mediated immune responses. Previously, we have shown that contact hypersensitivity (CHS), a cell-mediated cutaneous immune response in reaction to haptens, could be subclassified based on whether the hapten primed for Th1 or Th2 cytokines in cells isolated from draining lymph nodes. We also found that tolerance to a Th2-priming hapten could be induced only by simultane blockade of the CD40-CD40L and B7-CD28 at the time of sensitization. Here we demonstrate that blockade of CD40-CD40L signaling alone induces long-lasting unresponsiveness to the Th1 hapten 2,4-dinitrofluorobenzene (DNFB), and inhibits antigen-specific T cell proliferation in vitro. We find that CD40-CD40L signaling is required in the sensitization but not elicitation phase of DNFB-induced CHS, as treatment of mice with anti-CD40L monoclonal antibody (mAb) does not affect the response to hapten challenge in previously sensitized and untreated animals. Examination of cytokine production shows that anti-CD40L mAb decreases interferon-gamma production by draining lymph node cells from DNFB-sensitized mice, and reciprocally increases interleukin (IL)-4 production. Consistent with this Th1 to Th2 immune deviation, anti-CD40L mAb prevents the induction of IL-12 mRNA in regional lymph nodes, an event which is normally seen within 12 h following hapten sensitization. In contrast, suppression of CHS by CTLA4Ig decreased the production of all cytokines by draining lymph node cells. Together, these data show that blockade of the CD40-CD40L pathway by itself is sufficient to induce tolerance to DNFB-induced CHS, and that this is associated with blockade of IL-12 induction and Th1 to Th2 immune deviation.     

Lysophosphatidylcholine upregulates CD40 ligand expression in newly activated human CD4+ T cells

Lysophosphatidylcholine (lyso-PC) accumulates in tissues undergoing inflammation and atherosclerosis, where an infiltration of T cells is also seen. We found that lyso-PC increased IFN-gamma production and CD40L expression in CD4+ T cells stimulated with anti-CD3 Ab and recombinant CD80 molecules, whereas lyso-PC did not affect IL-2 and IL-4 production. These results suggest that lyso-PC, in combination with other stimuli, may regulate CD4+ T cell functions to propagate local inflammatory reactions and also imply a novel role played by a modified lipid in the selection of Th1/Th2 immune response as well as in the T cell mediated pathogenesis in atherosclerosis.     

CD4+ T cells inhibit growth of Epstein-Barr virus-transformed B cells through CD95-CD95 ligand-mediated apoptosis

Greater than 90% of the human population acquire Epstein-Barr virus (EBV) in infancy and retain a lifelong latent infection without any clinical consequences. Nevertheless EBV has been identified as the causal agent of infectious mononucleosis, and is associated with several tumours including endemic Burkitt's lymphoma and B cell lymphomas in immunosupressed patients. B cells infected with EBV are transformed in vitro and grow continuously as lymphoblastoid cell lines. The growth of EBV-transformed B cells in vivo is controlled by the immune system. Studies on immunity to EBV have mainly focused on MHC class I-restricted CD8+ cytotoxic T cells specific for viral latent antigens. Here it is reported that in vitro stimulation of peripheral blood lymphocytes by autologous EBV-infected B cells, which have been induced to express lytic cycle antigens, gives rise to a predominantly CD4+ T cell response. Furthermore, the growth of EBV-infected B cells can also be regulated by these activated CD4+ T cells through apoptosis mediated by CD95-CD95 ligand (CD95L). CD95-CD95L-mediated apoptosis is an important mechanism of normal B cell growth regulation. As EBV-transformed B cells remain susceptible to this mechanism, the control of EBV in vivo may be not only by virus-specific CD8+ cytotoxic T cell immunity but also by normal mechanisms of immune regulation of B cell growth.     

CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells

CD40 ligand (CD40L, CD154), a transmembrane protein structurally related to the cytokine TNF-alpha, was originally identified on stimulated CD4+ T cells, and later on stimulated mast cells and basophils. Interaction of CD40L on T cells with CD40 on B cells is of paramount importance for the development and function of the humoral immune system. CD40 is not only constitutively present on B cells, but it is also found on monocytes, macrophages and endothelial cells, suggesting that CD40L has a broader function in vivo. We now report that platelets express CD40L within seconds of activation in vitro and in the process of thrombus formation in vivo. Like TNF-alpha and interleukin-1, CD40L on platelets induces endothelial cells to secrete chemokines and to express adhesion molecules, thereby generating signals for the recruitment and extravasation of leukocytes at the site of injury. Our results indicate that platelets are not only involved in haemostasis but that they also directly initiate an inflammatory response of the vessel wall.     

IL-12 up-regulates CD40 ligand (CD154) expression on human T cells

IL-12 is a heterodimeric cytokine produced by APC that promotes the development of CD4+ Th1 cells and their IFN-gamma production after TCR/CD3 triggering. We here investigated the capacity of IL-12 to modify the expression on T cells of CD40 ligand (CD40L or CD154), a molecule transiently expressed on activated T cells and known to be of utmost importance for cognate interaction with B cells and for activation of dendritic cells and macrophages. Our data demonstrate that IL-12 up-regulates CD40L expression on anti-CD3-activated human peripheral blood T cells. For optimal induction of CD40L, IL-12 synergizes with IL-2 as well as with other costimulatory interactions, such as B7/CD28. The effect of IL-12 was observed at both the protein and the mRNA level. T cells costimulated by IL-12 provided more efficient help for IL-4-dependent B cell proliferation and for IgG production than when activated in the absence of IL-12. This helper activity was blocked by an mAb against CD40L, indicating that the effect of IL-12 on B cells is mediated indirectly through CD40L. The data thus suggest that the effects of IL-12 on cellular and humoral immune responses are partly mediated through CD40L induction.     

Immune regulatory loops determine productive interactions within human T lymphocyte-dendritic cell clusters

Help for the induction of cytolytic T lymphocytes is mediated by dendritic cells (DC) that are conditioned by CD40 signaling. We identified tumor necrosis factor family member CD27L/CD70, which is expressed by cytolytic T lymphocytes on interaction with DC to control CD154 (CD40L) up-regulation on CD45RA+ helper T cells for subsequent DC stimulation. The results show that the initiation of a cytolytic immune response is determined by regulatory circuits, requiring simultaneous activation and differentiation of all cells involved in T lymphocyte-DC cluster formation.     

Interleukin-12 release by mitogen-stimulated mononuclear cells in the elderly

Defects involving cellular expression of activation molecules, cell mediated immune response and natural killer (NK) activity are commonly observed in the elderly. Herein, data are reported on the evaluation of IL-12 production by old subjects. IL-12 is, actually, considered the key molecule for the induction of a T helper 1 (Th1) -type and NK response. IL-12 production from old subjects peripheral blood mononuclear cells (PBMNC) was evaluated using T-independent (bacterial lipopolysaccharide, LPS) or -dependent (phytoemagglutinin, PHA; immobilized anti-CD3 monoclonal antibodies, anti-CD3) mitogens. The IL-12 production after LPS stimulation was not reduced in cultures from old subjects when compared to that from young ones. On the contrary, IL-12 production by PHA or anti-CD3 stimulated PBMNC from old subjects was decreased. Furthermore, we have demonstrated a reduced CD40 and CD40 ligand (CD40L) expression on PBMNC from old subjects. This finding fits very well with the reduced cytokine production observed in the T-dependent stimulation systems, being the CD40-CD40L interaction mandatory for an efficient IL-12 production. All together, these results seem to suggest that defects in cell expression of activation molecules can affect the IL-12 secretion and in consequence other Th1-type cytokines.     

Hyphomycetes in morning dew of meadows

CD80 and CD86 (B7-1 and B7-2) are the ligands on antigen-presenting cells (APCs) which bind CD28 and deliver the costimulatory signals necessary for T cell activation. The reasons for the existence of two CD28 binding molecules are not well understood. We created a mutant version of CTLA4-Ig that could selectively bind CD80 and block CD28-CD80 interaction but leave CD28-CD86 binding intact. CD80 blockade prevented antigen-induced accumulation of eosinophils and lymphocytes in the lung of immunized mice, but did not block antigen induced systemic blood eosinophilia or IgE antibody production. No preferential expression of CD80 could be demonstrated on a population of lung APC consisting mainly of macrophages. These results indicate that CD80 costimulation is not necessary for the induction of Th2 immune responses but rather for the maintenance or amplification of lung inflammatory responses.     

Immunostimulatory effects of a plasmid expressing CD40 ligand (CD154) on gene immunization

Interaction of CD40 with its ligand (CD154) can induce CD40-bearing APCs to express immune stimulatory accessory molecules that facilitate immune recognition. We evaluated whether a plasmid vector encoding CD154 (pCD40L) could influence the immune response to a transgene protein encoded by coinjected plasmid DNA. We found that coinjection of pCD40L in BALB/c mice enhanced the Ab response to beta-galactosidase induced by i.m. or intradermal injection of placZ, a plasmid DNA vector encoding beta-galactosidase. Furthermore, i.m. or intradermal coinjection of pCD40L with placZ enhanced the generation of CTL specific for P815 cells transfected with placZ. This study indicates that pCD40L can serve as a genetic adjuvant capable of augmenting humoral and cellular immune responses to Ags encoded by plasmid DNA expression vectors.     

Alzheimer's beta-amyloid peptides induce inflammatory cascade in human vascular cells: the roles of cytokines and CD40

Accumulating evidence suggests that beta-amyloid (Abeta)-induced inflammatory reactions may partially drive the pathogenesis of Alzheimer's disease (AD). Recent data also implicate similar inflammatory processes in cerebral amyloid angiopathy (CAA). To evaluate the roles of Abeta in the inflammatory processes in vascular tissues, we have tested the ability of Abeta to trigger inflammatory responses in cultured human vascular cells. We found that stimulation with Abeta dose-dependently increased the expression of CD40, and secretion of interferon-gamma (IFN-gamma) and interleukin-1beta (IL-1beta) in endothelial cells. Abeta also induced expression of IFN-gamma receptor (IFN-gammaR) both in endothelial and smooth muscle cells. Characterization of the Abeta-induced inflammatory responses in the vascular cells showed that the ligation of CD40 further increased cytokine production and/or the expression of IFN-gammaR. Moreover, IL-1beta and IFN-gamma synergistically increased the Abeta-induced expression of CD40 and IFN-gammaR. We have recently found that Abeta induces expression of adhesion molecules, and that cytokine production and interaction of CD40-CD40 ligand (CD40L) further increase the Abeta-induced expression of adhesion molecules in these same cells. These results suggest that Abeta can function as an inflammatory stimulator to activate vascular cells and induces an auto-amplified inflammatory molecular cascade, through interactions among adhesion molecules, CD40-CD40L and cytokines. Additionally, Abeta1-42, the more pathologic form of Abeta, induces much stronger effects in endothelial cells than in smooth muscle cells, while the reverse is true for Abeta1-40. Collectively, these findings support the hypothesis that the Abeta-induced inflammatory responses in vascular cells may play a significant role in the pathogenesis of CAA and AD.     

Functional characterization of porcine CD4+CD8+ extrathymic T lymphocytes

The porcine immune system is unique in that the expression of CD4 and CD8 antigens defines four subpopulations of resting, extrathymic (CD1-) T lymphocytes. In addition to CD4-CD8+ and CD4+CD8- T lymphocytes, CD4-CD8- and CD4+CD8+ lymphocyte subpopulations are prominent in blood as well as in lymphoid tissues. In the present study, a functional comparison was made between CD4+CD8- and CD4+CD8+ T lymphocyte subpopulations. In a primary in vitro immune response against alloantigenic stimulator cells, both subpopulations proliferated without significant differences in their reactivity. Different results were obtained when analyzing the antigen-specific functions of the two CD4+ subpopulations in a secondary response against recall viral antigen; these experiments were performed with T lymphocytes from pseudorabies virus-immunized pigs. The proliferative response against viral antigens could be assigned to the CD4+CD8+ subpopulation, whereas the CD4+CD8- subpopulation remained nonreactive. Further analyses of the virus-specific in vitro immune response revealed a major histocompatibility complex (MHC) class II restricted helper T lymphocyte reaction involving CD4 but not CD8 molecules as restriction elements. Taken together, these results demonstrate that only the extrathymic CD4+CD8+ T lymphocyte subpopulation of swine contains MHC class II-restricted antigen-specific memory T helper cells.     

Acquired CD40-ligand deficiency in chronic lymphocytic leukemia

Patients with B-cell chronic lymphocytic leukemia (CLL) acquire an immunodeficiency with many characteristics similar to those of persons with inherited defects in the gene encoding the CD40-ligand (CD154). We found that the blood and splenic CD4+ T cells of patients with CLL failed to express surface CD154 after CD3 ligation. However, using an enzyme-linked immunosorbent assay (ELISA)-based quantitative competitive polymerase chain reaction (PCR), we noted that CD3 ligation could induce such T cells to express CD154 messenger RNA at levels similar to that of CD3-activated T cells from normal donors. Moreover, addition of increasing numbers of CLL B cells to activated normal donor T cells rapidly resulted in progressively greater down-modulation of CD154. Such down-modulation of CD154 could be blocked by addition of CD40 monoclonal antibody to cultures in vitro. We propose that leukemia cell-mediated down-modulation of CD154 on activated T cells accounts for some of the acquired immune defects of patients with CLL.     

Differential requirements for CD28 and CD40 ligand in the induction of experimental autoimmune myasthenia gravis

The interactions of CD28-B7 and CD40-CD40 ligand (CD40L) pathways in T cell costimulation and autoimmune disease are incompletely understood. We sought to address this issue by investigation of the genesis of acetylcholine receptor (AChR)-induced antibody-mediated experimental autoimmune myasthenia gravis (EAMG) in CD28- and CD40L-deficient mice (CD28-/-, CD40L-/-). Compared to wild-type mice, the CD28-/- mice became less susceptible, and CD40L-/- mice were completely resistant to EAMG induction. Analysis of T helper functions, reflected by cytokine responses, revealed a switch to a Th1 profile in CD28-/- mice. Consistently, levels of serum AChR-specific antibodies of the IgG1 isotype were decreased in CD28-/- mice. In the CD40L-/- mice, both Th1 and Th2 cytokine responses were diminished, and T cell-dependent AChR-reactive B cell responses were more severely impaired than in the CD28-/- mice. Thus, CD28 and CD40L are differentially required for induction of EAMG.     

CD40 expressed on thymic epithelial cells provides costimulation for proliferation but not for apoptosis of human thymocytes

Human thymic epithelial cells express CD40, so we examined the possible role of CD40 in activation of thymocytes. We observed that both CD4+CD8- and CD4-CD8+ thymocytes proliferate after stimulation by anti-CD3 mAb in the presence of cultured thymic epithelial cells. Costimulation of CD4+ thymocytes by thymic epithelial cells is partly inhibited by an anti-CD40 mAb, but this mAb has no effect on costimulation of CD8+ thymocytes. The selective costimulatory ability of CD40 for CD4+ thymocytes was confirmed in experiments in which thymocytes were stimulated with anti-CD3 in the presence of murine P815 cells transfected with CD40 cDNA. The level of costimulation induced by P815-CD40 was comparable with that induced by P815 cells expressing CD80 (B7.1). Treatment of thymocytes with the Ca2+ ionophore ionomycin and the phorbol ester PMA or with anti-CD3 mAb resulted in up-regulation of the CD40 ligand, suggesting that this molecule is involved in CD40-mediated costimulation of human thymocytes. Costimulation of thymocytes by CD80 strongly increased anti-CD3-induced death of fetal thymocytes. In contrast, costimulation by CD40 did not increase anti-CD3-mediated apoptosis of these thymocytes. To confirm that CD40 does not affect anti-CD3-induced cell death, we established a variant of the Jurkat T leukemic cell line that constitutively expresses CD40L and analyzed the sensitivity of this cell line for activation-induced apoptosis. In contrast to CD80, CD40 failed to increase anti-CD3-mediated apoptosis in CD40L+ Jurkat cells, whereas both CD40 and CD80 strongly increased IL-2 production induced by anti-CD3. These findings suggest that costimulation by CD40 is involved in clonal expansion of CD4+ thymocytes but not in activation-induced cell death.     

Specific suppression of human CD4+ Th cell responses to pig MHC antigens by CD8+CD28- regulatory T cells

Evidence that T cells can down-regulate the immune response by producing or consuming certain cytokines or by lysing APCs or Th cells has been provided in various systems. However, the generation and characterization of suppressor T cell lines have met with limited success. Here we show that xenospecific suppressor T cells can be generated by in vitro stimulation of human T cells with pig APCs. Similar to allospecific suppressors, these xenospecific suppressor T cells carry the CD8+CD28- phenotype and react to MHC class I Ags expressed by the APCs used for priming. TCR spectratyping of T suppressor cells showed oligoclonal usage of TCR-Vbeta families, indicating that xenostimulation of CD8+CD28- T cells results in Ag-driven selection of a limited Vbeta repertoire. Xenospecific T suppressor cells prevent the up-regulation of CD154 molecules on the membrane of Th cells, inhibiting their ability to react against the immunizing MHC class II xenoantigens. The mechanism of this suppression, therefore, appears to be blockade of CD154/CD40 interaction required for efficient costimulation of activated T cells.     

CD40 engagement up-regulates cyclooxygenase-2 expression and prostaglandin E2 production in human lung fibroblasts

A newly emerging view of fibroblasts is that they are vital for initiating inflammation and respond to and direct the activities of leukocytes. Human fibroblasts can express CD40, an activation Ag the ligand of which is displayed by activated leukocytes. We demonstrate here that CD40 engagement on human lung fibroblasts dramatically increases proinflammatory PGE2 synthesis. This up-regulation is mediated through an induction of cyclooxygenase-2 (Cox-2) since Cox-2-selective inhibitors block the up-regulation. Western and Northern blot analyses demonstrated that Cox-2 protein and mRNA are dramatically increased in fibroblasts following CD40 engagement. We conclude that CD40 is a major pathway in human fibroblasts for the induction of Cox-2. There is intense interest in devising strategies for disruption of the CD40-CD40 ligand system to blunt inflammation. Such an intervention would be expected to attenuate the up-regulation of fibroblast Cox-2 and PGE2 production at the site of tissue injury.