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.

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.

Intracellular processing and transport of NH2-terminally truncated forms of a hemagglutinin-neuraminidase type II glycoprotein.

Six amino-terminal deletion mutants of the NH2-terminally anchored (type II orientation) hemagglutinin-neuraminidase (HN) protein of parainfluenza virus type 3 were expressed in tissue culture by recombinant SV-40 viruses. The mutations consisted of progressive deletions of the cytoplasmic domain and, in some cases, of the hydrophobic signal/anchor. Three activities were dissociated for the signal/anchor: membrane insertion, translocation, and anchoring/transport. HN protein lacking the entire cytoplasmic tail was inserted efficiently into the membrane of the endoplasmic reticulum but was translocated inefficiently into the lumen. However, the small amounts that were successfully translocated appeared to be processed subsequently in a manner indistinguishable from that of parental HN. Thus, the cytoplasmic domain was not required for maturation of this type II glycoprotein. Progressive deletions into the membrane anchor restored efficient translocation, indicating that the NH2-terminal 44 amino acids were fully dispensable for membrane insertion and translocation and that a 10-amino acid hydrophobic signal sequence was sufficient for both activities. These latter HN molecules appeared to be folded authentically as assayed by hemagglutination activity, reactivity with a conformation-specific antiserum, correct formation of intramolecular disulfide bonds, and homooligomerization. However, most (85-90%) of these molecules accumulated in the ER. This showed that folding and oligomerization into a biologically active form, which presumably represents a virion spike, occurs essentially to completion within that compartment but is not sufficient for efficient transport through the exocytotic pathway. Protein transport also appeared to depend on the structure of the membrane anchor. These latter mutants were not stably integrated in the membrane, and the small proportion (10-15%) that was processed through the exocytotic pathway was secreted. The maturation steps and some of the effects of mutations described here for a type II glycoprotein resemble previous observations for prototypic type I glycoproteins and are indicative of close similarities in these processes for proteins of both membrane orientations.

Cytokine and cytokine receptor genes 'captured' by viruses.

Viruses have historically been used as tools to understand basic cellular functions. Recently viruses have been shown to encode genes whose protein products function to modulate the host immune system. Viral immune modulators can now be exploited for the purpose of further understanding cellular immune phenomena. These studies promise to provide a rich source of information for the cellular immunologist, the basic virologist, and the clinic.

Shared resources between the neural and immune systems: semaphorins join the ranks.

The semaphorin family of molecules contains members known to deliver guidance cues to migrating axons during development. Semaphorins also have been identified on the surface of hematopoietic cells and, interestingly, in the genomes of certain lytic viruses. Recent studies indicate that semaphorins bind with high affinity to at least two different receptor families and are biologically active on immune cells as well as neuronal cells.

Current approaches to the development of vaccines effective against parainfluenza and respiratory syncytial viruses.

Vaccines against parainfluenza (PIV) and respiratory syncytial viruses (RSV) that are currently being developed include both live and subunit vaccines. Candidate live PIV vaccines that have been found to be attenuated and efficacious in rodents or primate models are (1) cold-adapted, temperature-sensitive mutants of PIV-type 3 that have been serially passaged at low temperature (20 degrees C) in simian kidney tissue culture; (2) protease-activation mutants (PIV-1-Sendai), which have mutations that decrease the cleavability of their F glycoprotein by host cell protease; (3) an animal virus, bovine PIV-3 virus, which is antigenically related to the human PIV-3 virus, and (4) vaccinia recombinant viruses bearing RSV or PIV-3 glycoproteins. Subunit RSV and PIV-3 viruses are being produced and evaluated as immunogens. A major concern with these vaccines is the possibility of disease potentiation following virus infection as occurred previously with formalin-inactivated measles and RSV vaccines. Studies indicate that PIV-3 and RSV glycoprotein vaccines are immunogenic and efficacious in animals but insufficient data exist to estimate their capacity to potentiate disease. However, since a cotton rat model is available to detect potentiated disease resulting from infection of cotton rats previously immunized with formalin-inactivated RSV vaccine, it is now possible to systematically evaluate new vaccines in experimental animals for disease potentiation before studies are initiated in humans. It is likely within the next several years that one or more of these PIV or RSV vaccines will be tested in humans for safety and immunogenicity.

One step ahead of the game: viral immunomodulatory molecules.

For decades cell biologists have relied on viruses to facilitate the study of complex cellular function. More recently, the tragedy of the AIDS epidemic has focused considerable human and financial resources on both virology and immunology, resulting in the generation of new information relating these disciplines. As the miracle of the mammalian immune system unfolds in the laboratory, the elegance of the mechanisms used by co-evolving viruses to circumvent detection and destruction by the host becomes inescapably obvious. Although many observation of virus-induced phenomena that likely contribute to the virus's escape of immune surveillance are still empirical, many other such phenomena have now been defined at the molecular level and confirmed in in vivo models. Immune modulators encoded within viral genomes include proteins that regulate antigen presentation, function as cytokines or cytokine antagonists, inhibit apoptosis, and interrupt the complement cascade. The identification of such gene products and the elucidation of their function have substantially strengthened our understanding of specific virus-host interactions and, unexpectedly, have contributed to the recognition of potent synergy between viruses, which can result in an unpredictable exacerbation of disease in co-infected individuals. Because many viral immune modulators clearly have host counterparts, viruses provide a valuable method for studying normal immune mechanisms. It is conceivable that an improved understanding of virus-encoded immunomodulators will enhance our ability to design reagents for use in therapeutic intervention in disease and in vaccine development.

Sequence analysis of the P and C protein genes of human parainfluenza virus type 3: patterns of amino acid sequence homology among paramyxovirus proteins.

The complete nucleotide sequence of the P + C mRNA of human parainfluenza virus type 3 (PF3) was determined by sequencing cDNA, viral genomic RNA and mRNA. The P + C mRNA is 2009 nucleotides in length, exclusive of poly(A), and contains two overlapping open reading frames (ORFs). The P + C mRNA encodes two proteins, the 602 amino acid nucleocapsid phosphoprotein P and the 199 amino acid non-structural protein C. Peptide mapping confirmed that the two proteins are unrelated. Hybrid-arrest translation experiments assigned each of the two proteins to its respective ORF. These studies showed that the coding strategy of the PF3 P + C mRNA is similar to that of Sendai virus. Amino acid sequence alignment showed that the P and C proteins of PF3 and Sendai virus represent homologous pairs. However, these homologies are represented by high contents of accepted amino acid substitutions and by similarity in hydropathy profiles rather than by high contents of exact amino acid matches. Homology with the P and C proteins of measles, canine distemper and respiratory syncytial viruses was at the threshold of significance. The patterns of amino acid sequence homology among the paramyxovirus HN, F, NP, P and C proteins are compared.

Human parainfluenza virus type 3: messenger RNAs, polypeptide coding assignments, intergenic sequences, and genetic map.

cDNA clones of mRNAs for the major nucleocapsid protein (NP), the nucleocapsid P protein plus the nonstructural C protein (P+C), and the matrix protein (M) of human parainfluenza virus type 3 (PF3) were identified by hybrid arrest and hybrid selection of in vitro translation. Previously, cDNA clones were identified and sequenced for the hemagglutinin-neuraminidase glycoprotein (HN) and the fusion glycoprotein (F) mRNAs (N. Elango, J. E. Coligan, R. C. Jambou, and S. Venkatesan, J. Virol. 57:481-489, 1986; M. K. Spriggs, R. A. Olmsted, S. Venkatesan, J. E. Coligan, and P. L. Collins, Virology 152:241-251, 1986). Synthetic oligonucleotides, designed from nucleotide sequences of the cDNAs, were used to direct dideoxynucleotide sequencing of gene junctions in PF3 genomic RNA (vRNA). From sequencing of vRNA, a sixth viral gene was detected and identified as the large nucleocapsid protein (L) gene by hybridization of a synthetic oligonucleotide to intracellular PF3 mRNAs separated by gel electrophoresis. The order of the six PF3 genes on vRNA was 3'-NP-P+C-M-F-HN-L-5'. The five intergenic regions consisted of the trinucleotide 3'-GAA. The PF3 genes initiated with semiconserved 10-nucleotide gene-start sequences and terminated with semiconserved 12-nucleotide gene-end sequences. The M gene terminated with an aberrant gene-end sequence; analysis of intracellular mRNA showed that this aberrant sequence correlated with a disproportionately high accumulation of readthrough mRNA. These studies showed that PF3 encodes six unique mRNAs (NP, P+C, M, F, HN, and L) that encode seven proteins (NP, P, C, M, F, HN, and L) and provided evidence of a close relationship between PF3 and Sendai (murine parainfluenza type 1) viruses.

Sequence analysis of the matrix protein gene of human parainfluenza virus type 3: extensive sequence homology among paramyxoviruses.

The sequences of the human parainfluenza virus type 3 (PIV3) matrix (M) mRNA [1150 nucleotides exclusive of poly(A)] and predicted M protein (353 amino acids) were determined by sequence analysis of cloned cDNA and viral genomic RNA. The gene-end sequence of the M gene differed from the semi-conserved gene-end sequence of the other PIV3 genes by an apparent insertion of eight nucleotides. The PIV3 M protein shared high sequence homology with Sendai virus and moderate homology with measles virus and canine distemper virus. Statistical analysis of the available sequences showed that the M protein was the most highly conserved parainfluenza viral protein.

Immunization with vaccinia virus recombinants that express the surface glycoproteins of human parainfluenza virus type 3 (PIV3) protects patas monkeys against PIV3 infection.

Patas monkeys (Eryphrocebus patas) were immunized intradermally with two vaccinia virus recombinants that individually express the hemagglutinin-neuraminidase glycoprotein or the fusion glycoprotein of human parainfluenza virus type 3 (PIV3). These immunizations induced a high titer of PIV3 serum-neutralizing antibodies. At 1 month after immunization, monkeys were challenged intratracheally with PIV3. Subsequent virus replication was reduced in these monkeys by 3.2 log10 and 1.9 log10 (mean peak virus titers) in the upper and lower respiratory tracts, respectively, compared with control animals. The average duration of virus shedding was also reduced from 9.0 to 3.4 days in the upper respiratory tract and from 5.3 to 1.2 days in the lower respiratory tract. These findings demonstrate that a single intradermal dose of live recombinant vaccinia viruses can significantly restrict the replication of a virus which primarily infects the epithelial cells of the respiratory tract.

Fusion glycoprotein of human parainfluenza virus type 3: nucleotide sequence of the gene, direct identification of the cleavage-activation site, and comparison with other paramyxoviruses.

The complete sequences of the fusion (F) mRNA and protein of human parainfluenza virus type 3 (PF3) were determined from overlapping cDNA clones. To confirm the cDNA sequence, the complete sequence of the F gene was determined independently by dideoxynucleotide sequencing of genomic RNA using synthetic oligonucleotide primers. The mRNA contains 1845 nucleotides, exclusive of poly (A), has an unusually long (193-nucleotide) 5' nontranslated region, and encodes an F0 protein of 539 amino acids. The site within F0 of the proteolytic cleavage that activates fusion activity was established by direct amino acid sequencing of the NH2 terminus of the F1 subunit. The PF3 F0 protein shares major structural features with the previously sequenced F0 proteins of Sendai virus (murine parainfluenza type 1) and simian virus 5 (SV5, canine parainfluenza type 2), including: similarity in overall length; similarity in location of the site of the activating proteolytic cleavage; the presence of an NH2-terminal signal peptide and COOH-proximal membrane anchor; strong conservation of the sequence at the NH2 terminus of the F1 subunit; and nearly exact conservation in the number and positions of cysteine residues. Alignment of the F0 protein sequences of PF3 with those of Sendai, SV5, and respiratory syncytial virus (RSV) using a matrix that scores both amino acid matches and mismatches provided highly significant statistical evidence that all four proteins are related. The order of decreasing relatedness to PF3 was found to be: Sendai virus, SV5, and RSV.

Human B cell proliferation and Ig secretion induced by recombinant CD40 ligand are modulated by soluble cytokines.

Recombinant human CD40 ligand (hCD40L) was expressed on the surface of CV1/EBNA cells and examined for its ability to induce proliferation and Ig secretion from human B cells in the presence or absence of soluble cytokines. hCD40L was directly mitogenic in a dose-dependent fashion for purified tonsil B cells with maximal proliferation occurring at days 5 to 7. Proliferation induced by CD40L was significantly enhanced in the presence of IL-2, IL-4, or IL-10 and strongly suppressed by transforming growth factor-beta. Although IL-5, TNF-alpha, and IFN-gamma had no stimulatory effect in the presence of hCD40L alone, if IL-4 was also present in cultures, these cytokines enhanced the proliferative response above that seen with IL-4 alone. Interestingly, in the absence of IL-4, IFN gamma had an inhibitory effect on hCD40L-induced proliferation. Although CD40L alone did not enhance Ig secretion, addition of IL-2 or IL-10 to the cultures significantly elevated the levels of IgM, IgG1, and IgA that were observed. Addition of IL-4 to the cultures did not enhance secretion of these isotypes but had a weak inhibitory effect. However, CD40L-mediated induction of IgG4 and IgE was dependent on the presence of IL-4. Of the cytokines examined, only IL-10 enhanced IgE secretion under these conditions. Although transforming growth factor-beta only partially inhibited secretion of IgM, IgG1, and IgA, it was strongly suppressive for IgG4 and IgE production. Our data demonstrate that proliferation and Ig secretion induced in the presence of CD40L can be modulated in a positive and negative fashion by soluble cytokines. IL-2 and IL-10 specifically enhance IgM, IgG1, and IgA production although IL-4, despite costimulating B cell proliferation, does not augment secretion of these isotypes but provided an essential cosignal with CD40L for the production of IgG4 and IgE.

CD40 ligand and its role in X-linked hyper-IgM syndrome.

CD40 ligand (CD40L) on activated T cells binding to CD40 on B cells is of critical importance for Ig heavy-chain switching and rescue of B cells from apoptosis after somatic mutation in the germinal centre. Mutations in the CD40L gene are now known to cause X-linked hyper-IgM syndrome (HIGM1), an immunodeficiency characterized by the absence of serum IgG, IgA and IgE. In this review, we discuss how basic and clinical immunology have combined to provide major insights into the function of CD40 in T-B cell collaboration.

The G glycoprotein of human respiratory syncytial viruses of subgroups A and B: extensive sequence divergence between antigenically related proteins.

Two major antigenic subgroups (designated A and B) have been described for human respiratory syncytial virus (RSV). Previously, on the basis of reactivity patterns with monoclonal antibodies, the greatest intersubgroup variation was shown to occur in the G protein, the putative attachment glycoprotein. To delineate the molecular basis for this variation, we have determined the nucleotide and deduced amino acid sequences of the G mRNAs and proteins representing a subgroup A (Long strain) and a subgroup B (18537 strain) virus. These sequences were compared to the available G mRNA sequence for another subgroup A (A2 strain) virus. The Long G protein shared 94% amino acid identity with the A2 G protein. In contrast, the 18537 G protein shared only 53% amino acid identity with the A2 sequence; interestingly, most of the sequence divergence occurred in the proposed extracellular domain of the G protein. This extensive divergence for the G protein was significantly greater than that observed for other RSV proteins. Despite this considerable divarication, the proposed extracellular domains of the G proteins contained a single region of highly conserved sequence and secondary structure that may represent a conserved structural or function domain, perhaps involved in attachment to cellular receptors. Furthermore, this conserved region may comprise part of an epitope that is shared between the two subgroup G proteins and may significantly contribute to the fact that, despite extensive overall amino acid sequence divergence, the RSV G proteins maintain significant antigenic relatedness.

Structural characteristics of CD40 ligand that determine biological function.

CD40 ligand (CD40L) is a 33 kDa type II glycoprotein which is transiently expressed on the surface of T cells following activation. The demonstration that signals delivered by CD40L are essential for the process of affinity maturation and immunoglobulin isotype switching following antigenic challenge came from the study of X-linked hyper-IgM patients whose T cells cannot express functional CD40L. While some of the biological activities of CD40L, especially on B cells, can be mimicked by monoclonal antibodies (MAb) specific for CD40, it is becoming increasingly clear that CD40L also mediates various functional effects on other cell types. Not only are there distinctions between the activities of CD40L and CD40 MAb, but the manner in which CD40 is ligated appears to play an important part in the biological outcome of signaling through this receptor. In this review, we compare and contrast the activities which can currently be ascribed to CD40L and CD40 MAb and consider the role that ligand oligomerization plays in CD40-mediated signal transduction.