Sustained cytokine production and immunophenotypic changes in human neuroblastoma cell lines transduced with a human gamma interferon vector.

The majority of human neuroblastomas express low to undetectable levels of major histocompatibility complex (MHC) class I and II antigens (MHC-I and -II). We studied the effects of gamma interferon (gamma-IFN) transduction on expression of these antigens in six human neuroblastoma cell lines with and without genomic amplification of the N-myc oncogene. All six were stably transduced with an MoMLV-based gamma-IFN retroviral vector (DAh gamma-IFN). G418-resistant cells were assayed for MHC-I, MHC-II, B7-1, and neuroblastoma-associated antigen expression, as well as for gamma-IFN levels in cell culture supernatants. Sustained gamma-IFN production, 2 to > 1000 units/10(6) cells/d, was attained for five of six transduced cell lines and persisted for up to 9 months. This resulted in marked upregulation of MHC-I and MHC-II expression in LA-N-1, LA-N-6, and CHLA-127 cells and moderate upregulation in SK-N-Fi and SK-N-AS cells. One cell line (LA-N-1) had marked induction of MHC-I and MHC-II despite marginal levels of gamma-IFN production. Expression of CD28 ligand B7-1 (as determined by BB1 antibody) remained unchanged in all gamma-IFN-transduced cell lines tested. Expression of several neuroblastoma-associated antigens (NKH1A, 126-4, HSAN 1.2, HNK, 459, and 390) was upregulated in some of the gamma-IFN-transduced cell lines. These results demonstrate that preparation of gamma-IFN expressing neuroblastoma cells for immunotherapeutic purposes is feasible and that gamma-IFN transduction results in phenotypic changes that may improve immunogenicity of human neuroblastoma cells.

Lack of expression from a retroviral vector after transduction of murine hematopoietic stem cells is associated with methylation in vivo.

We describe studies of gene transfer and expression of the human glucocerebrosidase cDNA by a Moloney murine leukemia virus (MoMuLV)-based retroviral vector in a murine gene transfer/bone marrow transplant (BMT) model. Pluripotent hematopoietic stem cells (HSCs) were assayed as the colony-forming units, spleen (CFU-S) generated after serial transplantation. Transcriptional expression from the MoMuLV long-terminal repeat (LTR) was detected at a high level in the primary (1 degree) CFU-S and tissues of reconstituted BMT recipients. However, we observed transcriptional inactivity of the proviral MoMuLV-LTR in > 90% of the secondary (2 degrees) CFU-S and in 100% of the tertiary (3 degrees) CFU-S examined. We have compared the methylation status of the provirus in the 1 degree CFU-S, which show strong vector expression, to that of the transcriptionally inactive provirus in the 2 degrees and 3 degrees CFU-S by Southern blot analysis using the methylation-sensitive restriction enzyme Sma I. The studies demonstrated a 3- to 4-fold increase in methylation of the Sma I site in the proviral LTR of 2 degrees and 3 degrees CFU-S compared to the transcriptionally active 1 degree CFU-S. These observations may have important implications for future clinical applications of retroviral-mediated gene transfer into HSCs, where persistent gene expression would be needed for an enduring therapeutic effect.

Quantitative and functional studies of impaired natural killer (NK) cells in patients with myelofibrosis, essential thrombocythemia, and polycythemia vera. I. A potential role for platelet-derived growth factor in defective NK cytotoxicity.

In view of the possibility that immunological dysfunctions may be involved in initiating or contributing to the pathogenesis of myeloproliferative disease, we investigated quantitative and functional activity of natural killer (NK) cells in patients with polycythemia vera, essential thrombocythemia and myelofibrosis, and have demonstrated, especially in myelofibrosis, a measurable cytotoxic defect in the ability of their peripheral blood mononuclear cells to efficiently kill the standard NK target, K-562. Furthermore, highly purified, FACS-sorted CD16+ lymphoid cells from patients with myelofibrosis were consistently defective in their ability to lyse K-562 targets, and could not be augmented substantially with recombinant interleukin (IL)-2. Only patients with myelofibrosis had significantly lower percentages and absolute numbers of CD16+ cells as compared to patients with essential thrombocythemia and polycythemia vera. Further experiments demonstrated that patients with myelofibrosis, although having fewer CD16+ NK cells, had a significantly increased proportion of CD16+ cells with detectable platelet-derived growth factor (PDGF) on their surface. In contrast, surface PDGF was barely detectable on CD16+ cells from patients with polycythemia vera and essential thrombocythemia, as well as from normal controls. Having previously reported that physiologic quantities of PDGF significantly inhibit human NK cell cytotoxicity, and that patients with myelofibrosis and essential thrombocythemia have significantly elevated circulating levels of plasma PDGF, we now have demonstrated that pretreatment of normal NK cells with concentrated, PDGF-containing, platelet-poor plasma from patients with these diseases significantly inhibits NK cytotoxicity. This inhibitory effect was reversed by neutralization of plasma PDGF with anti-PDGF (coupled to Sepharose resin). Both the NK defects demonstrated in this study, and the abnormal plasma PDGF results reported earlier, are most striking in myelofibrosis and least abnormal in polycythemia vera, with an intermediate degree of abnormality in essential thrombocythemia. Our new findings suggest a causal correlation between abnormal platelet release, plasma accumulation of PDGF, and the observed NK immunodeficiency in these myeloproliferative patients.

Inhibition of human natural killer cell activity by platelet-derived growth factor (PDGF). III. Membrane binding studies and differential biological effect of recombinant PDGF isoforms.

We have previously reported that platelet-derived growth factor (PDGF) substantially inhibits human natural killer (NK) cell cytotoxicity, and that NK cells possess high-affinity surface binding sites for the PDGF-AB isoform. In this communication, we present direct evidence for the presence of A-type (alpha) PDGF receptors on human NK cells by demonstrating that human NK cells have approximately 150,000 high-affinity, surface binding sites for recombinant (r)PDGF-AA and approximately 300,000 high-affinity, surface binding sites for rPDGF-BB. This was determined by the competitive binding of 125I-labelled rPDGF-AA or 125I-labelled rPDGF-BB and homologous unlabelled rPDGF-AA or rPDGF-BB to FACS-sorted, CD16+ lymphoid (NK) cells, and Scatchard analysis of these data. In addition, we also demonstrate that the various isoforms of PDGF have differential effects on NK-cell cytotoxicity. Physiological quantities (100 ng/ml) of rPDGF-BB homodimers, highly purified PDGF-AB heterodimers from outdated platelets, and rPDGF-AB heterodimers substantially inhibited NK-cell cytotoxicity in both a dose- and time-dependent manner. In contrast, pretreatment of NK cells with equivalent nanogram amounts of rPDGF-AA homodimers resulted in a significantly weaker inhibitory effect on NK-cell cytotoxicity as compared with the PDGF-BB and PDGF-AB isoforms. The implications of these findings are discussed.

Multiple modifications in cis elements of the long terminal repeat of retroviral vectors lead to increased expression and decreased DNA methylation in embryonic carcinoma cells.

Infection by murine retroviruses in embryonic carcinoma (EC) and embryonic stem cells is highly restricted. The transcriptional unit of the Moloney murine leukemic virus (MoMuLV) long terminal repeat (LTR) is inactive in EC and embryonic stem cells in association with increased proviral methylation. In this study, expression in F9 EC cells was achieved from novel retroviral vectors containing three modifications in the MoMuLV-based retroviral vector: presence of the myeloproliferative sarcoma virus LTR, substitution of the primer binding site, and either deletion of a negative control region at the 5' end of the LTR or insertion of a demethylating sequence. We conclude that inhibition of expression from the MoMuLV LTR in EC cells is mediated through the additive effects of multiple cis-acting elements affecting the state of methylation of the provirus.

Cells expressing human glucocerebrosidase from a retroviral vector repopulate macrophages and central nervous system microglia after murine bone marrow transplantation.

Gaucher disease is an inherited lysosomal storage disease in which the loss in functional activity of glucocerebrosidase (GC) results in the storage of its lipid substrate in cells of the macrophage lineage. A gene therapy approach involving retroviral transduction of autologous bone marrow (BM) followed by transplantation has been recently approved for clinical trial. Amelioration of the disease symptoms may depend on the replacement of diseased macrophages with incoming cells expressing human GC; however, the processes of donor cell engraftment and vector gene expression have not been addressed at the cellular level in relevant tissues. Therefore, we undertook a comprehensive immunohistologic study of macrophage and microglia replacement after murine BM transplantation with retrovirus-marked BM. Serial quantitative PCR analyses were employed to provide an overview of the time course of engraftment of vector-marked cells in a panel of tissues. Following reconstitution of hematopoietic tissues with vector-marked donor cells at early stages, GC+ cells began to infiltrate the liver, lung, brain, and spinal cord by 3 months after transplant. Immunohistochemical analyses of PCR+ tissues using the 8E4 monoclonal antibody specific for human GC revealed that macrophages expressing human GC had partially reconstituted the Mac-1+ population in all tissues in a manner characteristic to each tissue type. In the brain, 20% of the total microglia had been replaced with donor cells expressing GC by 3 to 4 months after transplant. The finding that significant numbers of donor cells expressing a retroviral gene product immigrate to the central nervous system suggests that gene therapy for neuronopathic forms of lysosomal storage diseases as well as antiviral gene therapy for AIDS may be feasible.

In vivo properties of three human HER2/neu-expressing murine cell lines in immunocompetent mice.

BACKGROUND AND PURPOSE: Expression of the HER2/neu proto-oncogene, a receptor-like transmembrane protein expressed at low levels on some normal cells, is markedly increased in a subset of human breast, colon, lung, and ovarian cancers. A humanized HER2/neu antibody has been tested as a therapeutic agent in several clinical trials, with promising results. We have developed a family of anti-HER2/neu fusion proteins. To evaluate the immunologic efficacy of these proteins, it is critical that tumors expressing the target antigen can grow in immunologically intact mice. METHOD: To produce murine tumors expressing human HER2/neu on the surface, CT26, MC38, and EL4 murine cell lines were transduced by use of a retroviral construct containing the cDNA encoding the human HER2/neu gene. RESULTS: Histologic features and kinetics of tumor growth in subcutaneous space of the human HER2/neu-expressing cells were similar to those of the respective parental cell lines. Intravenous inoculation with these cells induced disseminated malignant disease. Flow cytometric and immmunohistochemical analyses of freshly isolated tumors revealed in vivo expression of human HER2/neu. Secretion of antigen was not detected by use of an ELISA. CONCLUSION: Although an antibody response against the human HER2/neu antigen was observed, this response does not affect the growth rate of the HER2/neu-expressing cells. These murine models may be useful tools for evaluation of anti-cancer therapeutic approaches that target human HER2/neu.