CD40 expression by human monocytes: regulation by cytokines and activation of monocytes by the ligand for CD40.

CD40 is a member of the tumor necrosis factor (TNF) receptor family of cell surface proteins and was originally described as a B cell restricted antigen. Treatment of primary human monocytes with granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), or interferon gamma (IFN-gamma) resulted in the induction of CD40 mRNA and enhancement of cell surface protein expression. CD40 was found to mediate monocyte adhesion to cells expressing recombinant CD40 ligand. CD40 ligand-transfected cells provided a potent costimulus for monocyte TNF-alpha and IL-6 production in the presence of GM-CSF, IL-3, or IFN-gamma, and enhanced IL-8 production stimulated by GM-CSF or IL-3. In addition, CD40 ligand-transfected cells acting in the absence of a costimulus induced monocytes to become tumoricidal against a human melanoma cell target. Collectively, these data indicate that CD40 ligand is pleiotropic with potent biological activity on monocytes.

Fas transduces activation signals in normal human T lymphocytes.

The Fas gene encodes a cell surface molecule that is a member of the the nerve growth factor/tumor necrosis factor receptor family of proteins and can mediate programmed cell death (apoptosis) in certain transformed cell lines. To characterize further the biological function of Fas, particularly with regard to its function in normal cells, a panel of monoclonal antibodies (mAbs) was generated against the extracellular portion of human Fas. Some of these mAbs induced apoptosis in transformed cell lines expressing Fas, but only when immobilized on the culture vessel. One of the new Fas mAbs (M38) was used for studies on normal lymphoid cells and found to stimulate the proliferation of purified human T cells and thymocytes when immobilized on culture wells along with CD3 antibody. T cell proliferation induced by Fas mAb was largely interleukin 2 independent and was demonstrated to be due to a direct effect on the precursor T cell. Thus, the data demonstrate that in addition to a role in the induction of apoptosis in certain transformed cell lines, the Fas protein may also play an important role in the activation and proliferation of normal T cells.

Recombinant human CD40 ligand stimulates B cell proliferation and immunoglobulin E secretion.

Signaling through the cell surface molecule, CD40, is known to play an important role in the proliferation and differentiation of B lymphocytes. Using the thymoma cell line EL4, we recently identified and cloned a cDNA encoding a murine ligand for the CD40 molecule (mCD40-L) and showed that it has biological activity in vitro. A cDNA encoding a human homologue of the mCD40-L was isolated using crosshybridization techniques from an activated peripheral blood T cell library. The predicted amino acid sequence indicates that this human ligand for CD40 (hCD40-L) is a 261 amino acid type II membrane protein that exhibits 78% amino acid identity with its murine counterpart. Northern blot and FACS analyses suggest that the hCD40-L is restricted in its expression to T lymphocytes, and that it is most abundant on the CD4+ T cell subpopulation. Cells transfected with hCD40-L caused the proliferation of human tonsil B cells in the absence of costimuli and, in the presence of interleukin 4, induced immunoglobulin E secretion from purified human B cells. A comparison of the efficacy of the hCD40-L and mCD40-L in these assays is presented.

Humoral immune responses in CD40 ligand-deficient mice.

Individuals with X-linked hyper-IgM syndrome fail to express functional CD40 ligand (CD40L) and, as a consequence, are incapable of mounting protective antibody responses to opportunistic bacterial infections. To address the role of CD40L in humoral immunity, we created, through homologous recombination, mice deficient in CD40L expression. These mice exhibited no gross developmental deficiencies or health abnormalities and contained normal percentages of B and T cell subpopulations. CD40L-deficient mice did display selective deficiencies in humoral immunity; basal serum isotype levels were significantly lower than observed in normal mice, and IgE was undetectable. Furthermore, the CD40L-deficient mice failed to mount secondary antigen-specific responses to immunization with a thymus-dependent antigen, trinitrophenol-conjugated keyhole limpet hemocyanin (TNP-KLH). By contrast, the CD40L-deficient mice produced antigen-specific antibody of all isotypes except IgE in response to the thymus-independent antigen, DNP-Ficoll. These results underscore the requirement of CD40L for T cell-dependent antibody responses. Moreover, Ig class switching to isotypes other than IgE can occur in vivo in the absence of CD40L, supporting the notion that alternative B cell signaling pathways regulate responses to thymus-independent antigens.

CD40 ligand gene defects responsible for X-linked hyper-IgM syndrome.

The ligand for CD40 (CD40L) is a membrane glycoprotein on activated T cells that induces B cell proliferation and immunoglobulin secretion. Abnormalities in the CD40L gene were associated with an X-linked immunodeficiency in humans [hyper-IgM (immunoglobulin M) syndrome]. This disease is characterized by elevated concentrations of serum IgM and decreased amounts of all other isotypes. CD40L complementary DNAs from three of four patients with this syndrome contained distinct point mutations. Recombinant expression of two of the mutant CD40L complementary DNAs resulted in proteins incapable of binding to CD40 and unable to induce proliferation or IgE secretion from normal B cells. Activated T cells from the four affected patients failed to express wild-type CD40L, although their B cells responded normally to wild-type CD40L. Thus, these CD40L defects lead to a T cell abnormality that results in the failure of patient B cells to undergo immunoglobulin class switching.

Tumor necrosis factor receptor superfamily members and their ligands.

The tumor necrosis factor (TNF) receptor family comprises a number of type I integral membrane glycoproteins which exhibit sequence homology in their cysteine-rich extracellular domains. Recently, ligands for many of these receptors have been identified. These molecules all display sequence identity with TNF and lymphotoxin beta, prototype ligands for this receptor family, and have the conformation of type II transmembrane molecules. While certain biological activities are common to many members of this TNF-like family, other activities appear to be shared only by some ligands, or are unique. The identification of the TNF superfamily of ligands has provided the opportunity to compare and contrast the diverse biological effects mediated through the interaction of these related molecules with their receptors.

B-cell stimulation.

Recent studies have identified CD40 ligand (CD40L) as the critical membrane-expressed molecule responsible for T cell dependent B-cell activation. CD40L co-operates with various cytokines to induce B-cell activation, proliferation, and immunoglobulin isotype switching. Some antigens, however, can also stimulate B-cell activation and isotype switching in the absence of CD40L or T cells. Recent studies have suggested that cytokines derived from non-T cells, such as natural killer cells, macrophages and mast cells, are responsible for isotype switching in T cell independent responses.

Repression of apoptosis in human B-lymphoma cells by CD40-ligand and Bcl-2: relationship to the cell-cycle and role of the retinoblastoma protein.

Using a Burkitt lymphoma cell line to model human B-cell apoptosis in vitro, we observed that crosslinking, by antibody, of cell surface immunoglobulin induced G1 growth-arrest followed by apoptosis. By contrast, cells treated with the Ca(2+)-ionophore, ionomycin, generated apoptotic signals in G2/M as well as in G1. Both ionomycin and anti-immunoglobulin treatment induced rapid dephosphorylation of Rb prior to apoptosis. Apoptosis was repressed following exposure to CD40-ligand and was accompanied by hyperphosphorylation of Rb and cell-cycle progression but not Bcl-2 expression. Expression of Bcl-2 protein in stable bcl-2-transfectants, also resulted in repression of apoptosis and anti-immunoglobulin-treated cells no longer underwent growth-arrest. In Bcl-2-expressing cells in which apoptosis was repressed, Rb remained hyperphosphorylated, even during G1-arrest induced by ionomycin. TGF beta treatment of Bcl-2-expressing cells induced G1-arrest, de-phosphorylation of Rb and apoptosis. These results suggest that the functional activity of Bcl-2 in B-lymphoma cells is dependent upon, or leads to, sustained hyperphosphorylation of Rb and that Rb hyperphosphorylation can be uncoupled from cell-cycle progression.

Differential effects of BCL-2 on survival and proliferation of human B-lymphoma cells following gamma-irradiation.

Bcl-2 can inhibit apoptosis induced by a variety of stimuli, including radiation and its presence in tumour cells would be expected to indicate poor prognosis. Bcl-2-expressing tumours, however, are often low-grade and highly responsive to therapy. To investigate this apparent paradox, we analysed in vitro the responses of Burkitt lymphoma (BL) cells to gamma-irradiation in the presence and absence of Bcl-2. High-level expression of Bcl-2 was shown to promote BL cell survival following irradiation. However, a significant proportion of Bcl-2-rescued cells subsequently underwent apoptosis after an extended period in culture. In addition, in different BL lines, Bcl-2 was found either to promote or to inhibit long-term proliferative activity following gamma-irradiation. This differential regulation of proliferation correlated both with differential effects of Bcl-2 on the cell cycle and with differences in p53 status. Thus, by one week after irradiation, BL cells expressing only wild-type p53 (wt/wt) had arrested in G1, whereas those with a mutant allele (wt/mu) were arrested in all phases of the cell cycle. The proportion of Bcl-2-rescued cells that subsequently underwent apoptosis was reduced by ligation of CD40 at the time of irradiation in wt/wt BL cells, but not in wt/mu cells. CD40-ligation reduced both G1-arrest and apoptosis in parallel. These results indicate that, whilst Bcl-2 can delay apoptosis in BL cells following gamma-irradiation, the protein can also cause growth-arrest and thereby promote apoptosis. Long-term survival following Bcl-2-mediated rescue of gamma-irradiated cells may depend on p53 status and require additional death-repressing or growth-promoting signals.

CD40-induced growth inhibition in epithelial cells is mimicked by Epstein-Barr Virus-encoded LMP1: involvement of TRAF3 as a common mediator.

CD40, a member of the tumour necrosis factor receptor family, is expressed on the surface of B lymphocytes where its ligation provides a potent survival signal. CD40 is also expressed in basal epithelial cells and in a number of different carcinomas where its function remains unknown. We observed that contrary to the studies in normal B cells, CD40 ligation in carcinoma cell lines and in normal primary epithelial cells resulted in growth inhibition and enhanced susceptibility to apoptosis induced by anti-neoplastic drugs, TNF-alpha, Fas and ceramide. This effect was also observed in CD40-transfected Rat-1 fibroblasts. The expression of Bcl-2 did not affect growth inhibition induced by CD40 ligation in epithelial cells but the Epstein - Barr Virus-encoded latent membrane protein 1 (LMP1) blocked the effect. Whilst transient expression of LMP-1 resulted in the inhibition of epithelial cell growth, this effect was not observed with a LMP1 mutant lacking the binding domain for TRAF3, a protein which may mediate signal transduction by interacting with the cytoplasmic domains of both CD40 and LMP1. Transient expression of TRAF3 also inhibited epithelial cell growth, whilst expression of a dominant-negative TRAF3 partially blocked the inhibitory effect of CD40 ligation and of transient LMP1 expression. These results suggest that CD40 regulates epithelial cell growth in a manner mimicked by LMP1 and implicate TRAF3 as a common mediator in the transduction of the growth inhibitory signals generated via the CD40 and LMP1 pathways.

CD40 ligand, Bcl-2, and Bcl-xL spare group I Burkitt lymphoma cells from CD77-directed killing via Verotoxin-1 B chain but fail to protect against the holotoxin.

Owing to its lineage and differentiation stage-restricted expression, CD77 has been mooted as a therapeutic target in Burkitt lymphoma (BL). The recognition that the globotriaosyl moiety of this neutral glycosphingolipid is a receptor for Escherichia coli-derived Verotoxin-1 (Shiga-Like Toxin-1) offers a potential delivery system for the attack. Here we show that CD77-expressing Group I BL cells which are normally susceptible to activation-induced death on binding Verotoxin-1 B chain are protected in the presence of CD40 ligand. Ectopic expression of either bcl-2 or bcl-xL also afforded resistance to the actions of the B chain. In total contrast, neither of the survival genes nor a CD40 signal - even when acting in concert - protected against killing mediated by the holotoxin. These findings indicate that while therapeutic modalities for CD77-expressing B cell tumors (which include follicular lymphoma) based on the use of Verotoxin-1 B chain might be compromised by the activation of endogenous or exogenous survival pathways, those exploiting the holotoxin should be left unscathed.

Phase I study of recombinant human CD40 ligand in cancer patients.

PURPOSE: To determine the toxicity, maximum-tolerated dose (MTD), and pharmacokinetics of recombinant human CD40 ligand (rhuCD40L) (Avrend; Immunex Corp, Seattle, WA), suggested in preclinical studies to mediate cytotoxicity against CD40-expressing tumors and immune stimulation. PATIENTS AND METHODS: Patients with advanced solid tumors or intermediate- or high-grade non-Hodgkin's lymphoma (NHL) received rhuCD40L subcutaneously daily for 5 days in a phase I dose-escalation study. Subsequent courses were given until disease progression. RESULTS: Thirty-two patients received rhuCD40L at three dose levels. A total of 65 courses were administered. The MTD was 0.1 mg/kg/d based on dose-related but transient elevations of serum liver transaminases. Grade 3 or 4 transaminase elevations occurred in 14%, 28%, and 57% of patients treated at 0.05, 0.10, and 0.15 mg/kg/d, respectively. Other toxicities were mild to moderate. At the MTD, the half-life of rhuCD40L was calculated at 24.8 +/- 22.8 hours. Two patients (6%) had a partial response on study (one patient with laryngeal carcinoma and one with NHL). For the patient with laryngeal cancer, a partial response was sustained for 12 months before the patient was taken off therapy and observed on no additional therapy. Three months later, the patient was found to have a complete response and remains biopsy-proven free of disease at 24 months. Twelve patients (38%) had stable disease after one course, which was sustained in four patients through four courses. CONCLUSION: The MTD of rhuCD40L when administered subcutaneously daily for 5 days was defined by transient serum elevations in hepatic transaminases. Encouraging antitumor activity, including a long-term complete remission, was observed. Phase II studies are warranted.

Modelling the human immune response: can mice be trusted? Commentary.

The mouse is now the animal of choice for laboratory-based medical research. Although its contribution to advancing understanding of our inner workings is indisputable, we should acknowledge that mice and humans are tangibly different. This article highlights, and attempts some rationale for, discrepancies between the two species' immune systems.

VH repertoire in human B lymphocytes stimulated by CD40 ligand and IL-4: evidence for positive and negative selection mechanisms coupled to CD40 activation.

In the normal immune system, B cells are thought to be negatively or positively selected at various checkpoints during their maturation; a process that maintains a broad immunoglobulin repertoire while eliminating non-functional or potentially harmful autoreactive antibodies. This study tested the hypothesis that utilization of certain immunoglobulin heavy chain variable region (VH) genes, possibly as a consequence of intrinsic affinity for various ligands, directs positive or negative B cell selection coupled to B cell activation in the periphery during the immune response. The specific prediction that the VH repertoire of CD40-activated B cells would differ from the repertoire of unstimulated cells from the same donor, was tested by assessing VH utilization among human B cell clones grown in vitro, following stimulation with CD40 ligand (CD40L) and IL-4. The results showed that, although utilization of the known VH families and of individual VH3 genes was similar to that found in unstimulated B lymphocytes of the same donor, utilization of individual VH4 genes in CD40-activated B cells displayed a pattern that was markedly different from that of the unstimulated B cells. An allele of V4-61, V4-61b, was over-represented among the activated cells and, in contrast, the V4-34 gene (known to encode cold agglutinins with strong autoreactive properties) was modestly represented among the VH4 activated B cells, although V4-34 was overwhelmingly predominant in the repertoire of resting B cells. These results point to the existence of selection mechanisms that operate during B cell activation in the periphery. These mechanisms may favor B cells utilizing certain VH genes and disfavor the cells that utilize other genes, possibly because utilization of the latter confers autoreactivity.

Bone marrow macrophages and megakaryocytes express common acute lymphoblastic leukaemia antigen.

Common acute lymphoblastic leukaemia antigen (CALLA) was demonstrated on a proportion of bone marrow macrophages and megakaryocytes. CALLA was detected by two monoclonal antibodies (J5 & BA3) in a three-layer immunoalkaline phosphatase system applied to routine air-dried bone marrow smears. The J5 staining was confirmed by an indirect immunofluorescent method and the CALLA was shown to be at the surface of the macrophages and megakaryocytes by an indirect immunogold technique. The findings are discussed in relation to the known tissue distribution of CALLA and to the clinical use of anti-CALLA antibodies for bone marrow purging.

Lymphoid cells, small cell lung cancer cells and epithelial cells share a membrane determinant which effects cell proliferation.

Two monoclonal antibodies (mAb) 1D1 and 2A6 were obtained from a fusion following hyperimmunization with prolymphocytic leukaemia (PLL) B cells. These mAb stain a minority of B- and T-cell leukaemias and approximately 20% of peripheral blood and tonsil T and B cells, activated with a variety of mitogens. Interestingly, all small cell lung cancer (SCLC) and bladder carcinoma lines examined were also stained by both mAb. On sections of normal and malignant tissue 1D1 and 2A6 show strong but distinct reactivity with epithelium, and in the case of ID1 staining is also present on endothelial tissue. The addition of purified 1D1 and 2A6 to Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines (B-LCL) and SCLC lines caused a significant increase in the rate of proliferation of these cells. Capping experiments have suggested that these two mAb, despite showing significantly different staining profiles, probably recognize distinct epitopes of the same surface molecule. These studies confirm that a lymphoid-cell associated antigen(s) detected by mAbs 1D1 and 2A6 is expressed on a wide range of normal and malignant cells and related cell lines.

Expression of receptors for interleukin 4 and interleukin 7 on human T cells.

Human recombinant interleukin 4 (IL-4) and interleukin 7 (IL-7) have been modified with biotin-N-hydroxysuccinimide and used to examine the expression of human IL-4 and IL-7 receptors (R) on activated peripheral blood T cells by flow cytometry. Freshly isolated T cells expressed only a low level of IL-4R which remained unchanged when cells were cultured in the absence of stimuli. In the presence of IL-4, IL-7, phytohemagglutinin A (PHA) or immobilized CD3 monoclonal antibody the intensity of biotinylated IL-4 staining increased approximately twofold on the majority of cells. A combination of mitogen with either IL-4 or IL-7 caused a considerable increase in IL-4 receptor expression over that seen in the presence of mitogen alone. IL-2 alone failed to induce IL-4R although it was able to cause a significant increase in receptor expression on T cells co-cultured with PHA or CD3. Freshly isolated T cells expressed high levels of IL-7R, as determined by biotinylated IL-7 binding and flow cytometry, which did not change significantly with culture in medium alone. Stimulation with PHA, Concanavalin A (Con A) or CD3 had little effect on the intensity of staining. In contrast, activation with phorbol ester resulted in a decrease in IL-7R expression. Similarly, in the presence of IL-4 or IL-7, but not IL-2, the intensity of staining with biotinylated IL-7 was lowered. Analysis of purified T-cell populations showed that IL-7R were present, and IL-4R could be induced, on both CD4+ and CD8+ populations. Analysis of IL-4 receptor expression by this flow cytometric technique was supported by results from 125I-labeled IL-4 binding and by Northern blot analysis of mRNA levels. Taken together, the results of these studies show that the use of biotinylated cytokines and flow cytometry provides a very sensitive method with which to study the expression and regulation of cytokine receptors.