Neuroblastoma cells expressing mature IL-18, but not proIL-18, induce a strong and immediate antitumor immune response.

Retroviral constructs were designed to express the novel cytokine interleukin 18 (IL-18), also known as interferon-gamma-inducing factor, in a murine neuroblastoma cell line [neuro-2a (N-2a)] to examine the effects of IL-18 expression on tumorigenicity. N-2a cells expressing proIL-18 (N-2a/IL-18p) were as tumorigenic as parental N-2a cells, whereas N-2a cells engineered to secrete mature IL-18 (N-2a/IL-18m) were nontumorigenic. Inoculation of mice with N-2a/IL-18m generated immediate immunity to parental N-2a. N-2a/IL-18m formed tumors in mice depleted of CD4+ and CD8+ T cells, suggesting that the antitumor immune response was T cell mediated. The resulting T-helper (Th) immune response was also characterized in vitro and had a large Th1 component based on in vitro production of the cytokines IFN-gamma and granulocyte macrophage colony-stimulating factor in response to tumor cells and IL-18.

Retrovirus-mediated gene transfer of B7-1 and MHC class II converts a poorly immunogenic neuroblastoma into a highly immunogenic one.

The T cell co-stimulatory molecule B7-1 was transduced into a poorly immunogenic murine neuroblastoma cell line (Neuro-2a, N-2a) alone or in combination with MHC class II genes to test the ability of these genes to stimulate antitumor immunity. N-2a cells transduced with B7-1 exhibited reduced tumorigenicity, whereas N-2a cells overexpressing both MHC class II (syngeneic, I-Ak) and B7-1 totally abrogated tumorigenicity. Rejection of I-Ak/B7-1 cells was dependent on both CD4+ and CD8+ T cells. The ability of both vaccines to induce protection against parental N-2a was temporally dependent on the time of secondary N-2a challenge. To investigate the immunity generated by N-2a/B7-1 and N-2a/I-Ak/B7-1 vaccines, we tested the ability of these modified cells to stimulate in vitro the proliferation of syngeneic splenocytes from naive mice. A significant increase in splenocyte proliferation was observed with N-2a/I-Ak/B7-1 cells compared to N-2a cells. We also determined that vaccination with N-2a/I-Ak/B7-1 cells was able to generate cytotoxic T cell responses to unmodified N-2a cells. The introduction of B7-1 and I-Ak into N-2a was able to convert a poorly immunogenic tumor to a highly immunogenic one; however, mice bearing large established unmodified tumors had little response to vaccination with N-2a/I-Ak/B7-1 cells. Our results emphasize the importance of tumor immunogenicity in the treatment of established tumors with MHC class II/B7-1 tumor cell vaccines.

Murine neuroblastoma vaccines produced by retroviral transfer of MHC class II genes.

Malignant tumors express tumor-related antigens, but effective antitumor immunity does not occur in the primary host. One hypothesis is that there is insufficient stimulation of T-cell responses due to ineffective antigen presentation. An approach to overcome these deficiencies is to modify tumor cells to express major histocompatibility complex (MHC) class II genes and thus facilitate the presentation of antigens directly by tumor cells. Our experiments with a murine neuroblastoma cell line (neuro-2a) transduced with DR (xenogeneic), 1-Ab (allogeneic), or 1-Ak (syngeneic) MHC class II genes support this notion. The relative potencies of the modified neuro-2a to induce immunity to unmodified neuro-2a were neuro-2a/DR > neuro-2a/1-Ab > neuro-2a/1-Ak. Modified neuro-2a also could stimulate naive splenocyte proliferation in vitro. The relative magnitude of the proliferative responses seen after stimulation with modified tumor cells was neuro-2a/DR > neuro-2a/1-Ab > neuro-2a/1-Ak > unmodified neuro-2a. Hence, the tumor cell-induced splenocyte proliferative responses observed in vitro correlate with the effectiveness of the tumor cell vaccines to induce antitumor immunity in vivo. These data show that the expression of exogenous MHC class II on tumor cells is a potent stimulus for specific antitumor immunity. Because of the correlation of the in vivo and in vitro immune responses to modified tumor cells, the tumor-induced lymphocyte proliferation assay may be useful in evaluating tumor cell vaccines produced by additional genetic modifications of tumor cells.

The Drosophila homeotic target gene centrosomin (cnn) encodes a novel centrosomal protein with leucine zippers and maps to a genomic region required for midgut morphogenesis.

The products of the homeotic genes in Drosophila are transcription factors that are necessary to impose regional identity along the anterior-posterior axis of the developing embryo. However, the target genes under homeotic regulation that control this developmental process are largely unknown. We have utilized an immunopurification method to clone target genes of the Antennapedia protein (ANTP). We present here the characterization of centrosomin (cnn), one of the target genes isolated using this approach. The spatial and temporal expression of the cnn gene in the developing visceral mesoderm (VM) of the midgut and the central nervous system (CNS) of wild-type and homeotic mutant embryos is consistent with the idea that cnn is a homeotic target. In the VM, Antp and abdominal-A (abd-A) negatively regulate cnn, while Ultrabithorax (Ubx) shows positive regulation. In the CNS, cnn is regulated positively by Antp and negatively by Ubx and abd-A. Characterization of a cDNA encoding CNN predicts a novel structural protein with three leucine zipper motifs and several coiled-coil domains exhibiting limited homology to the rod portion of myosin. Immunocytochemical results demonstrate that the cnn encoded protein is localized to the centrosome and the accumulation pattern is coupled to the nuclear and centrosome duplication cycles of cleavage. In addition, evidence suggests that the expression of the cnn gene in the VM correlates with the morphogenetic function of Ubx in that tissue, i.e., the formation of the second midgut construction. The centrosomal localization of CNN and the involvement of microtubules in midgut morphogenesis suggest that this protein may participate in mitotic spindle assembly and the mechanics of morphogenesis through an interaction with microtubules, either directly or indirectly.

Basic fibroblast growth factor promotes adhesive interactions of neuroepithelial cells from chick neural tube with extracellular matrix proteins in culture.

Fibroblast growth factors have been increasingly assigned mitogenic and trophic roles in embryonic and postnatal development of the nervous system. Little is known, however, of their functional roles in early embryonic neural development at the neural tube stage. We have examined the effect of basic fibroblast growth factor (bFGF) on the adhesive behavior in culture of dissociated brachio-thoracic neural tube cells from 26- to 30-somite stage chick embryos. Cells plated on collagen-coated substratum at a low density attach to the substratum but show poor cell spreading. Addition of bFGF markedly promotes cell spreading, yielding an epithelial morphology. This effect becomes discernible 6-8 hours after cell plating with bFGF and is completed by 24 hours, with half-maximal and maximal effects attained at around 0.4 and 10 ng/ml, respectively. The number of cells remain largely constant up to 24 hours, and then cell survival and/or mitogenic effects of bFGF become apparent. The cell spreading effect is abolished by cycloheximide treatment, inhibited by the anti-beta 1-integrin antibody CSAT, and accompanied by about twofold increases in the expression of beta 1-integrin and vinculin, components of focal adhesion complexes. Cells cultured with bFGF for 24 hours exhibit enhanced cell attachment and cell spreading with little time lag following cell plating. In earlier embryonic stages, developmentally less mature cells depend much more on bFGF for their cell spreading and survival, while in later stages the cell spreading response to bFGF becomes undetectable as neural tube develops to spinal cord. The cell spreading effect of bFGF is realized on specific extracellular matrix proteins including laminin, fibronectin and collagen, but not on vitronectin, arg-gly-asp peptide (PepTite-2000), poly-L-ornithine or others. These results suggest that, in an early stage of neural tube development, bFGF is involved in the developmental regulation of adhesive interactions between neuroepithelial cells and the extracellular matrix, thereby controlling their proliferation, migration and differentiation.

Homeotic genes have specific functional roles in the establishment of the Drosophila embryonic peripheral nervous system.

The Drosophila embryonic peripheral nervous system (PNS) contains segment-specific spatial patterns of sensory organs which derive from the ectoderm. Many studies have established that the homeotic genes of Drosophila control segment specific characteristics of the epidermis, and more recently these genes have also been shown to control gut morphogenesis through their expression in the visceral mesoderm (Tremml, G. and Bienz, M. (1989), EMBO J. 8, 2677-2685). We report here the roles of homeotic genes in establishing the spatial patterns of sensory organs in the embryonic PNS. The PNS was examined in embryos homozygous for mutations in the homeotic genes Sex combs reduced (Scr), Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A) and Abdominal-B (Abd-B) with antibodies that label specific subsets of sensory organs. Our results suggest that the homeotic genes have specific roles in establishing the correct spatial patterns of sensory organs in their normal domains of expression. In addition, we also report the effects of ectopic expression of the homeotic genes labial (lab), Deformed (Dfd), Scr, Antp or Ubx on the normal development of sensory organs in the embryonic PNS. Interestingly, while previous studies have concluded that ectopic expression of the homeotic genes Dfd, Scr and Antp has no effect on the segmental identity of the abdominal segments, our results demonstrate that this is not true. We show that ectopic expression of these genes does result in the disruption of the developing PNS in the abdomen. Our results are suggestive of a role for the homeotic gene products in regulating genes which are necessary for generating sensory progenitor cells in the developing PNS.

Expression of nerve growth factor (NGF) receptors in the developing inner ear of chick and rat.

The expression of nerve growth factor receptors (NGFRs) was studied in the developing inner ear with in situ hybridization in chick embryos and with immunocytochemistry in rat embryos to determine sites of possible functions of NGF or NGF-like molecules in inner ear development. NGFR expression in the chick otocyst and acoustic ganglion is compared with epithelial differentiation and the onset of afferent innervation as determined with fluorescent carbocyanine tracers. In the inner ear of the chick embryo, NGFR mRNA expression shows an alternating pattern in mesenchymal and epithelial tissues. NGFR mRNA is heavily expressed in the mesenchyme surrounding the otocyst (E2-3), ceases at E3-5, and reappears in a thin layer of mesenchymal cells surrounding the membraneous epithelia (E5-13). In the otocyst epithelium, NGFR mRNA expression develops in one anterior and one posterior focus at E3-4.5. NGFR mRNA is expressed in the primordia of the ampullary cristae (E5-7) and possibly the anlage of the utricle; label transiently concentrates in the planum semilunatum of the cristae ampullares and in superior portions of the semicircular canals at E9, but is not seen in differentiating hair cells. In the acoustic ganglion, NGFR mRNA expression begins at E4; at the same time, the first peripheral acoustic nerve processes penetrate the otic epithelium (E4-4.5). The acoustic ganglia remain weakly NGFR mRNA-labeled in the posthatch animal. In the rat embryo, NGFR immunoreactivity is present in the auditory placode at E9, in the periotic mesenchyme at E9-10, and in the medial half of the otocyst at E10-11. At E12, epithelial NGFR expression becomes restricted anteriorly and posteriorly in a pattern similar to that of the chick otocyst and ceases at E13. NGFR immunoreactivity appears transiently in pillar cells of the cochlea in the third week of gestation. NGFR and NGFR mRNA is expressed after E11 in the acoustic ganglia. While NGFR transcripts are expressed in the cochlear ganglion cell bodies, NGFR protein becomes restricted to neuronal processes by the third week of gestation. The vestibular, but not the cochlear (spiral) ganglia remain NGFR-labeled in the adult rat. Onset of NGFR mRNA expression in the acoustic ganglion during the period of afferent fiber ingrowth into the otocyst epithelium is consistent with the hypothesis that NGF-like molecules may have a neurotrophic function for acoustic ganglion cells. Transient expression of NGFRs in secretory cells of the vestibular endorgan and pillar cells in the organ of Corti implicate a role for neurotrophins in the differentiation of these epithelial cell types.

Expression of nerve growth factor (NGF) receptors in the brain and retina of chick embryos: comparison with cholinergic development.

The expression of nerve growth factor receptor (NGFR) transcripts was investigated with in situ hybridization techniques in the CNS of chick embryos from 3 days of incubation (E3) to 14 days posthatch (P14). The time course and distribution of NGFR expression was compared with the development of the cholinergic phenotype. Cholinergic properties were assessed by immunolabeling for choline acetyltransferase (ChAT) and histochemistry for acetylcholinesterase (AchE) activity. NGFR transcripts are expressed transiently in the inner plexiform layer and ganglion cell layer of the retina (E4-P1), neostriatum and hippocampus (E18), infundibular hypothalamus (E7-18), spiriform complex (E9-15), layers 2, 3 (E9-18), and 10 (E11-18) of the optic tectum, nucleus mesencephalicus profundus, pars ventralis (E9-18), parvicellular isthmic nucleus (E7-P1), magnocellular isthmic nucleus (E9-E18), nucleus semilunaris (E7-18), isthmo-optic nucleus (E7-P14), rostral motor nuclei (E5-18), developing cerebellum (E7-15), internal granule cell layer (E11-18) and Purkinje cell layer (E15-P14) of the cerebellar cortex, and the inferior olivary nucleus (E9-15). A small number of neuronal populations with embryonic expression of NGFR remain strongly NGFR-positive in the posthatch animal:habenular nuclei (labeled after E5), nucleus subrotundus (after E9), mesencephalic trigeminal nucleus (after E5), caudal parts of locus ceruleus and nucleus subceruleus (after E7), medullar reticular nuclei (after E11), and motor nuclei IX, X, and XII (after E9). The majority of neuronal populations with NGFR expression show cholinergic properties in development, and NGFR expression always precedes the onset of ChAT immunoreactivity. Postnatal expression of growth factor receptors is largely confined to neurons of the reticular type. NGFR expression in avian CNS nuclei differs from that in mammals. Early loss of NGFR expression in the cholinergic basal forebrain (which remains strongly NGFR positive in mammals) and persistent NGFR expression in parts of the avian locus ceruleus indicate changes of growth factor receptor expression and growth factor requirements in phylogeny. Knowledge of the time and distribution of NGFR expression in the chick embryo will facilitate the assessment of specific functions of NGF and NGF-like molecules in an embryonic model with easy access for experimental manipulations.(ABSTRACT TRUNCATED AT 400 WORDS)

Alternating phases of FGF receptor and NGF receptor expression in the developing chicken nervous system.

Patterns of expression of transcripts encoding receptors for fibroblast growth factor and nerve growth factor (FGF-R and NGF-R) in the developing chick nervous system are compared using in situ hybridization histochemistry. FGF-R transcripts are expressed abundantly in the germinal neuroepithelial layer. Expression ceases as cells migrate into the mantle layer and returns during late maturation of neuronal populations, including cholinergic nuclei of the basal forebrain, brainstem reticular and motor nuclei, and cerebellar Purkinje and granule neurons. The pattern of NGF-R expression is generally reciprocal to that of FGF-R in the CNS and in some phases of development of the PNS. These results suggest that FGF and NGF may act sequentially rather than in concert during neuronal development.

Structure and developmental expression of the chicken NGF receptor.

The nucleotide and deduced amino acid sequence of a cDNA clone of the chicken NGF receptor (NGFR) is reported and is compared with sequences of mammalian NGF receptors. A model is presented in which monodentate or bidentate binding of NGF dimers to repeated cysteine-rich sequence elements of the receptor yields low- or high-affinity NGF binding, respectively. In situ hybridization is used to characterize expression of NGFR in developing chick from 40 hr to 10 days of embryogenesis. NGFR mRNA expression is detected in premigratory neural crest cells, in epibranchial placode cells, and in all sensory, sympathetic and parasympathetic derivatives of these structures. In the embryonic CNS, NGFR mRNA is detected in the mantle zone but not the periventricular germinal zone throughout most of the neural tube. By Embryonic Day 8, NGFR mRNA is detected in a substantial fraction of cells in every brain region, with highest levels present in developing motor neurons. NGFR mRNA also is transiently expressed in many mesenchymal cell populations including cells in branchial arch, sclerotome, muscle anlagen, and feather follicles. The functional significance of wide-spread embryonic expression of the NGF receptor is discussed.