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.     

Demonstration of functional CD40 in B-lineage acute lymphoblastic leukemia cells in response to T-cell CD40 ligand

Because activated T cells were previously shown to induce proliferation of human normal B-cell precursors (BCP) via the CD40 pathway, we investigated the effects of T cells on leukemic blasts isolated from patients with B-lineage acute lymphoblastic leukemia (BCP-ALL). An anti-CD3 activated human CD4+ T-cell clone was found to induce significant call proliferation in four of nine BCP-ALL samples analyzed. In one of these cases, the T-cell effect was clearly dependent on interaction between CD40 and its ligand. Accordingly, a more thorough analysis was performed on this particular leukemia (case 461, adult early pre-B-ALL, mBCR+, Philadelphia+, i(9q)+). Thus, autologous CD4+ T cells isolated from the patient were also able to induce CD40-dependent proliferation of the leukemic blasts. Analysis of the phenotype after coculture showed that, among the CD19+ cells, a proportion gradually lost expression of CD10 and CD34, whereas some cells acquired CD23. In addition, and in contrast with normal BCP, activated T cells promoted maturation of a subset of the case 461 leukemic cells into surface IgM+ cells. The leukemic origin of the cycling and the maturing cells was assessed by the presence of i(9q), a chromosomal abnormality associated with this leukemia and evidenced by fluorescence in situ hybridization. Taken together, these results show that leukemic BCP can be activated via CD40 but that not all cases display detectable stimulation in response to T cells despite their expression of CD40. In addition, the present data suggest that CD4+ T cells could potentially play a role in the physiology of BCP-ALL.     

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.     

CD40/CD40 ligand interactions in normal, reactive and malignant lympho-hematopoietic tissues

CD40 is a 48 Kd integral membrane protein expressed by cells of B cells, origin, dentritic cells, monocytes, epithelial cells, endothelial cells and tumor cells including carcinomas, B cell lymphomas/leukemias and Hodgkin and Reed-Sternberg (HRS) cells of Hodgkin's disease (HD). CD40 has been clustered as a member of the nerve growth factor (NGF)/tumor necrosis factor (TNF) receptor superfamily with the corresponding counterstructure, the CD40 ligand (L) being mainly expressed by activated CD4+ T cells, but also some activated CD8+ T cells, basophils, eosinophils, mast cells and stromal cells. CD40L shares significant amino acid homology with TNF particularly in its extracellular domain ("TNF homology region") and is therefore viewed as a member of the TNF ligand superfamily. Binding of CD40L+ T cells to CD40+ B cells is thought to play a major role in T cell-dependent B cell activation, B cell proliferation, Ig isotype switching, memory B cell formation and rescue of B cells from apoptotic death in germinal centers. Mutations of the CD40L gene have been associated with the X-linked hyper-IgM immunodeficiency syndrome, pointing to the critical role of the CD40/CD40L interaction in the T cell-B cell interplay. Accordingly, expression of CD40 by human lympho-hematopoietic tumors has been shown in most of the B cell neoplasias, H-RS cells and HD and some carcinomas. In contrast, CD40L+ tumor cells are almost invariably restricted to CD4+/CD8- T cell lymphomas. Overall, functional CD40/CD40L interactions appear to be critical for cellular activation signals during immune responses and neoplastic tumor cell growth. The understanding of the biology of CD40L has improved our diagnostic and therapeutic repertoire in the management of several human diseases, including CD40+ tumors.     

CD40 ligand inhibits Fas/CD95-mediated apoptosis of human blood-derived dendritic cells

Dendritic cells (DC) are considered to be the most potent antigen-presenting cells (APC) in the immune system. In this study, we analyzed the regulation of apoptosis of human peripheral blood-derived DC. DC were generated from adherent peripheral blood mononuclear cells that had been cultured for 7 days with granulocyte-macrophage colony-stimulating factor and interleukin-4. These cells displayed phenotypic properties of DC, including dendritic processes, expression of CD1a and lack of expression of CD14, and were very potent at presenting soluble antigens to T cells. Blood-derived DC were demonstrated to express the Fas/CD95 antigen and an agonist antibody to CD95 strongly induced apoptotic cell death in these cells. Soluble trimeric CD40 ligand potently inhibited both CD95-mediated and spontaneous apoptosis in DC. The data suggest that interactions between members of the tumor necrosis factor family of ligands expressed by T cells with their receptors on DC play an important role in the regulation of apoptosis in DC during antigen presentation and may, therefore, regulate the duration of T cell expansion and cytokine production.     

Increase of intracellular calcium is the essential signal for the expression of CD40 ligand

CD40 ligand (CD40L) is present on activated but not on resting T cells. In contrast to the activation markers CD25 and CD71, a strong CD40L expression could be induced by calcium ionophore alone but not by phorbol 12-myristate 13-acetate (PMA). Ionomycin induced a very early mRNA and protein surface expression of CD40L within the first 2 h, whereas CD25 and CD71 did not appear earlier than 6 h after stimulation. The mitogens phytohemagglutinin and concanavalin A induced little CD40L, but together with PMA, a markedly increased CD40L expression was observed. In T cells stimulated with immobilized anti-CD3, co-stimulation with anti-CD28 or PMA induced an earlier and higher maximal CD40L expression. CD40L expression of purified T cells was higher and more prolonged compared to that of T cells in unseparated peripheral blood mononuclear cells. We conclude that the expression of CD40L on T cells is profoundly different from other early activation markers with regard to signal requirements, kinetics and the role of accessory cells in the system.     

Antitumor responses induced by transgenic expression of CD40 ligand

Because CD40 ligand (CD40L) is a co-stimulator molecule for multiple components of the immune response, we wanted to determine whether transgenic expression of the molecule would increase immune responses against a weakly immunogenic murine tumor, neuro-2a. Tumor cells were transduced with a retroviral construct containing the CD40L gene and co-injected with variable numbers of non-CD40L transduced cells into syngeneic mice. Mice injected with cells that expressed CD40L had a significant reduction in average tumor size as compared to controls (p < 0.0001). In addition, survival of the neuro-2a/CD40L mice was 48 days versus 34 days for the neuro-2a/neo controls (p < 0.02). Expression of CD40L by less than 1.5% of neuro-2a cells was sufficient for significant antitumor effects (p < 0.001). These antitumor effects protected mice from subsequent challenge with parental neuro-2a cells. The protective effects of CD40L were associated with systemic immunomodulation. In vivo depletion of CD8+ cells abrogated the CD40L-mediated antitumor effects. Analysis of spleens from CD40L-protected animals showed increased numbers of CD4+ and CD8+ cells, the majority of which co-expressed the activation marker CD25. In addition, an increased number of antigen-presenting cells (APCs) expressed the co-stimulatory molecule CD86. These experiments illustrate that transducing even a small percentage of tumor cells with CD40 ligand can create a long-lasting systemic immune response capable of impeding growth of unmodified neuroblastoma cells.     

CD40 ligand and autoantigen are involved in the pathogenesis of low-grade B-cell lymphomas of mucosa-associated lymphoid tissue

Low-grade MALT-type lymphomas are malignancies of mucosal marginal-zone B cells and preceded by reactive inflammatory lymphoid tissue. Experimental observations suggest that antigen and CD40 Ligand act during cognate T/B cell interaction and are crucial for germinal center B-cell maturation generating marginal-zone B cells. To investigate the mechanisms underlying the development of extranodal MALT-type lymphomas, the immunoglobulin receptor was sequenced and analyzed for antigen specificity using heterohybridoma technology. Furthermore, CD40 ligand expression was evaluated by immunohistochemistry and by semiquantitative RT-PCR, and ligand binding to the CD40 of tumor B cells was studied using the CD40 system. Hypermutations were found in low-grade lymphomas throughout CDR1-CDR3 suggestive of positive selection through their antigen receptor. Different VH families were used and more than 69% of tumor immunoglobulins bound different mucosal antigens. CD40L expression was found in the tumor marginal zone in substantial amounts. The in vitro proliferation response of all low-grade MALT-type lymphomas was dependent on anti-CD40-mediated signals and cytokines. Our data provide evidence that autoantigen as well as the CD40L expressed by activated nonneoplastic T cells may drive the evolution of low-grade MALT-type lymphomas either directly or by paracrine mechanisms and that antigen may contribute to lymphoma pathogenesis.     

CD40 ligand/CD40 stimulation regulates the production of IFN-gamma from human peripheral blood mononuclear cells in an IL-12- and/or CD28-dependent manner

CD40 ligand (CD40L)/CD40 costimulation is an important regulator of Th1 responses. Two mechanisms by which CD40L/CD40 stimulation may enhance IFN-gamma are via direct induction of IL-12 and augmentation of the expression of costimulatory molecules such as B7 from APCs. We examined the ability of CD40L/CD40 stimulation to regulate the production of IFN-gamma through IL-12 and/or CD28 costimulation from human PBMCs stimulated with T cell-specific stimuli. The roles of exogenous and endogenous CD40L/CD40 stimulation were evaluated using a trimeric soluble CD40L agonist (CD40T) and an anti-CD40L Ab, respectively. The presence of CD40T in cultures increased the production of IL-12 and IFN-gamma from PBMCs stimulated with varying amounts of PHA. The mechanism, however, by which CD40T enhanced IFN-gamma varied according to the level of T cell activation. Under maximal stimulatory conditions (PHA, 1/100), an IL-12-dependent pathway was dominant. At relatively low levels of T cell stimulation (PHA, 1/500 and 1/1000), however, an additional IL-12-independent CD28-dependent pathway was elucidated. We further studied the role of exogenous CD28 stimulation in regulating the production of IFN-gamma. The enhancement of IFN-gamma production induced by direct CD28 stimulation was primarily dependent on endogenous IL-12 or CD40L/CD40 stimulation. Together, these data suggest that the production of IFN-gamma involves a complex interaction between two interdependent, yet distinct, costimulatory pathways and provide evidence that CD40T may be an effective adjuvant for the enhancement of responses.     

CD40 and its ligand in host defense

The genetic changes involved in the metastatic process of ovarian epithelial cancer remain undetermined. The expression of nm23, a putative metastasis-suppressor gene product, was assessed immunohistochemically in malignant and benign ovarian neoplasms, considering histology of tumors and clinical advancement of disease. Comparison of nm23 protein content in tissue sections and respective cyst and/or ascitic fluid cells was also performed. Significant heterogeneity of nm23 immunostaining was observed, and no correlation with histological subtype of ovarian carcinoma was found. Expression of nm23 was higher in carcinomas compared with benign tumors. A significant trend to have a higher nm23 reactivity in ascitic fluid cells vs. primary tumors was observed. Our results indicate that the increase of nm23 reactivity is activated in the early stages of the disease and that the progression of ovarian carcinoma is accompanied by overexpression of nm23 protein. Our observations did not confirm the postulated role of nm23 as a suppressor gene in ovarian cancer.     

Apoptosis and expression of inducible nitric oxide synthase are mutually exclusive in renal mesangial cells

Nitric oxide (NO) is a multipurpose messenger molecule, important for blood vessel relaxation, neuronal communication, and antimicrobial activities. The generation of NO from L-arginine is catalyzed by NO synthase (NOS). An inducible form of NOS, iNOS, was first characterized in macrophages and then in many other tissues and cells, including renal mesangial cells. Mesangial cells play a crucial role in the regulation of the glomerular filtration rate as well as in the pathophysiology of certain forms of glomerulonephritis in which mesangial cells and macrophages produce NO in high amounts. Because reports have associated NO production with apoptotic cell death in macrophages and we recently demonstrated NO-mediated apoptosis in mesangial cells, we searched for the relationship between in situ iNOS induction and apoptosis by iNOS immunocytochemistry and terminal desoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining. RAW 264.7 macrophages exhibited homogeneous iNOS expression and apoptotic nuclei in the iNOS-containing cells upon stimulation with interferon-gamma and lipopolysaccharide. In contrast, stimulated rat mesangial cells stained heterogeneously for iNOS, depending on cell passage and iNOS-stimulating pathway. Mesangial cells expressing iNOS did not display signs of apoptosis and, vice versa, cells showing characteristic features of apoptosis did not stain for iNOS. Thus, our study suggests that mesangial cells react to stimulation by interleukin-1 and/or cAMP-elevating compounds with mutually exclusive responses, either by expression of iNOS or by undergoing programmed cell death.     

Chronic lymphocytic leukemia B cells can express CD40 ligand and demonstrate T-cell type costimulatory capacity

Chronic lymphocytic leukemia (CLL) is characterized by a clonal expansion of CD5(+) B cells in the peripheral blood. Associated immune aberrations include abnormal Th-cell function and pathogenic autoantibodies. Under most circumstances, CLL B cells do not proliferate in culture and express a limited repertoire of surface antigens, including CD19, CD20, CD23, CD27, CD40, and CD70. In this report, we demonstrate that freshly isolated B cells from a subset of CLL cases constitutively express CD40 ligand (CD40L, CD154), a member of the tumor necrosis factor family which is normally expressed by activated CD4(+) T cells and mediates T-cell-dependent B-cell proliferation and antibody production. The degree of CD40L expression varied considerably among the CLL cases examined. CD40L was detected in purified CLL B cells by immunofluorescence flow cytometry, by RT-PCR, and by immunoprecipitation. To demonstrate that CD40L in the CLL B cells is functional, we used irradiated CLL cells to stimulate IgG production by target, nonmalignant B cells in coculture. The CLL B cells induced IgG production by normal B cells to a similar degree as did purified T cells in a process which was partially inhibited by monoclonal antibody to CD40L. This is one of the first reports of CD40L expression in a B-cell tumor. The data suggest that CD40L in the tumor cells may be a factor in the generation of pathologic antibodies by normal B cells in some patients with CLL.     

A new death receptor 3 isoform: expression in human lymphoid cell lines and non-Hodgkin's lymphomas

Two isoforms encoding the full-length transmembrane death receptor 3 (DR3) were isolated from mRNAs of a panel of human cell lines and tumor tissues obtained from patients with follicular non-Hodgkin's lymphoma. A new DR3 variant (DR3 beta) was characterized by 2 insertions of respectively 20- and 7-base pairs (bp) which result in a predictive translated polypeptide differing from the described DR3 molecule by a 28 amino-acid stretch in the extracellular domain. DR3 was shown to be expressed in all cell lines and lymphoma samples tested, whereas DR3 beta expression was restricted to lymphoid T-cell and immature B-cell lines and to selected cases with follicular lymphoma. These data provide new insight into the molecular heterogeneity of DR3, suggesting the presence of several receptor isoforms that can participate in lymphoid cell homeostasis.     

Enhancement of antitumor immunity by expression of CD70 (CD27 ligand) or CD154 (CD40 ligand) costimulatory molecules in tumor cells

BACKGROUND: Most catalytic RNAs depend on divalent metal ions for folding and catalysis. A thorough structure-function analysis of catalytic RNA therefore requires the identification of the metal-ion-binding sites. Here, we probed the binding sites using Fenton chemistry, which makes use of the ability of Fe2+ to functionally or structurally replace Mg2+ at ion-binding sites and to generate short-lived and highly reactive hydroxyl radicals that can cleave nucleic acid and protein backbones in spatial proximity of these ion-binding sites. RESULTS: Incubation of group I intron RNA with Fe2+, sodium ascorbate and hydrogen peroxide yields distinctly cleaved regions that occur only in the correctly folded RNA in the presence of Mg2+ and can be competed by additional Mg2+, suggesting that Fe2+ and Mg2+ interact with the same sites. Cleaved regions in the catalytic core are conserved for three different group I introns, and there is good correlation between metal-ion-binding sites determined using our method and those determined using other techniques. In a model of the T4 phage-derived td intron, cleaved regions separated in the secondary structure come together in three-dimensional space to form several metal-ion-binding pockets. CONCLUSIONS: In contrast to structural probing with Fe2+/EDTA, cleavage with Fe2+ detects metal-ion-binding sites located primarily in the inside of the RNA. Essentially all metal-ion-binding pockets detected are formed by tertiary structure elements. Using this method, we confirmed proposed metal-ion-binding sites and identified new ones in group I intron RNAs. This approach should allow the localization of metal-ion-binding sites in RNAs of interest.     

Loss of p16/INK4A protein expression in non-Hodgkin's lymphomas is a frequent finding associated with tumor progression

The CDKN2A gene located on chromosome region 9p21 encodes the cyclin-dependent kinase-4 inhibitor p16/INK4A, a negative cell cycle regulator. We analyzed p16/INK4A expression in different types of non-Hodgkin's lymphoma to determine whether the absence of this protein is involved in lymphomagenesis, while also trying to characterize the genetic events underlying this p16/INK4A loss. To this end, we investigated the levels of p16/INK4A protein using immunohistochemical techniques in 153 cases of non-Hodgkin's lymphoma, using as reference the levels found in reactive lymphoid tissue. The existence of gene mutation, CpG island methylation, and allelic loss were investigated in a subset of 26 cases, using single-strand conformational polymorphism and direct sequencing, Southern Blot, polymerase chain reaction, and microsatellite analysis, respectively. Loss of p16/INK4A expression was detected in 41 of the 112 non-Hodgkin's lymphomas studied (37%), all of which corresponded to high-grade tumors. This loss of p16/INK4A was found more frequently in cases showing tumor progression from mucosa-associated lymphoid tissue low-grade lymphomas (31 of 37) or follicular lymphomas (4 of 4) into diffuse large B-cell lymphomas. Analysis of the status of the p16/INK4A gene showed different genetic alterations (methylation of the 5'-CpG island of the p16/INK4A gene, 6 of 23 cases; allelic loss at 9p21, 3 of 16 cases; and nonsense mutation, 1 of 26 cases). In all cases, these events were associated with loss of the p16/INK4A protein. No case that preserved protein expression contained any genetic change. Our results demonstrate that p16/INK4A loss of expression contributes to tumor progression in lymphomas. The most frequent genetic alterations found were 5'-CpG island methylation and allelic loss.     

WIN 35,428 and mazindol are mutually exclusive in binding to the cloned human dopamine transporter

It has been suggested that cocaine and mazindol bind to separate sites on the dopamine transporter. In the present study, we address this issue by examining the inhibition by mazindol of the binding of [3H]WIN 35,428 ([3H]2beta-carbomethyoxy-3beta-(4-fluorophenyl)-tropane), a phenyltropane analog of cocaine, and the inhibition by WIN 35,428 of [3H]mazindol binding to the cloned human dopamine transporter expressed in C6 glioma cells. The design involved the construction of inhibition curves at six widely different radioligand levels, enabling the distinction between the nonlinear hyperbolic competition (i.e., negative allosteric) model and the competitive (i.e., mutually exclusive binding) model. Nonlinear computer curve-fitting analysis indicated no difference in the goodness of fit between the two models; the negative allosteric model indicated an extremely high allosteric constant of approximately > or = 100, which practically equates to the competitive model. The present results suggest that complex interactions reported between cocaine and mazindol in inhibiting dopamine transport are beyond the level of ligand recognition.     

T-cell receptor BJ gene segment expression in human umbilical cord blood CD4+ and CD8+ T-cell subsets

By employing RT-PCR-based technology, followed by Southern-blot analysis, patterns of relative TRC BJ gene segment usage in human CD4+ and CD8+ umbilical cord blood T cells (UCT) from ten children were determined in relation to seven recombined TCR BV gene (sub) families (BV 3, 5S1, 6S1-3, 8, 9, 12 and 18). Normal frequency of usage of individual BJ members was observed to be extremely nonrandom. BJ usage in association with each BV was ranked and mean ranking values were calculated for individual BJs. Moreover, BJ family usage and family ranges as well as individual BJ over-representations were determined. In all these aspects of BJ exon expression, CD4+ and CD8+ UCT displayed similar distribution patterns. Comparisons of BJ usage in UCT subpopulations and in the adult peripheral blood lymphocyte (PBL) counterparts were performed and many similarities were observed. However, discrepancies in two parameters were recorded; contrary to observations in PBL, individual BJ over-representations were virtually absent in UCT, and significantly less wide BJ family ranges were demonstrated in CD8+ UCT relative to CD8+ PBL T cells. These differences support the notion that UCT are in a less dynamic state than are PBL T cells. Hence, despite the fact that PBL T cells are subjected to continuous antigenic challenge, the striking resemblance of PBL and UCT with regard to the overall individual relative usage, ranking, mean ranking and family utilisation of BJ gene segments, irrespective of the choice of recombined BV exons, may suggest a relatively nondiscriminatory role for the BJ gene product in antigen recognition as compared to those encoded by the BV, (N) and BD gene segments.