A novel DNA-binding regulatory factor is mutated in primary MHC class II deficiency (bare lymphocyte syndrome).

Regulation of MHC class II gene expression is an essential aspect of the control of the immune response. Primary MHC class II deficiency is a genetically heterogeneous disease of gene regulation that offers the unique opportunity of a genetic approach for the identification of the functionally relevant regulatory genes and factors. Most patients exhibit a characteristic defect in the binding of a nuclear complex, RFX, to the X box motif of MHC class II promoters. Genetic complementation of a B-lymphocyte cell line from such a patient with a cDNA expression library has allowed us to isolate RFX5, the regulatory gene responsible for the MHC class II deficiency. This gene encodes a novel DNA-binding protein that is indeed a subunit of the RFX complex. Mutations in the RFX5 gene have been characterized in two patients. Transfection of the patient's cells with the RFX5 cDNA repairs the binding defect and fully restores expression of all the endogenous MHC class II genes in vivo.

Coexpression of stem cell factor and its receptor c-Kit in human malignant glioma cell lines.

Stem cell factor (SCF), a hematopoietic growth factor, is the ligand of the tyrosine kinase receptor encoded by the c-kit proto-oncogene. Beside the important role of this receptor-ligand complex in hematopoiesis, gametogenesis and melanogenesis, SCF and its receptor have been shown to be expressed in the brain. We have studied the expression of SCF and c-kit in 20 human malignant glioma cell lines at the mRNA as well as at the protein level. In addition, recombinant human (rh) SCF was tested in [3H]thymidine uptake assays for a mitogenic effect on these cells. SCF and c-Kit proteins were detected in the cytoplasm of glioma cells by alkaline phosphatase-monoclonal anti-alkaline phosphatase immunostaining and Western blot analysis. However, neither SCF nor c-Kit were seen on the cell surface by flow cytometry. Furthermore, none of the proliferation assays showed a mitogenic effect for exogenously added rhSCF. Blocking studies using an anti-SCF antibody failed to demonstrate modulating effects on the growth of selected cell lines. These results suggest that SCF and c-Kit may mediate non-proliferative signals or may employ intracellular mechanisms for autocrine growth regulation of glioma cells.

Cystic fibrosis-type mutational analysis in the ATP-binding cassette transporter signature of human P-glycoprotein MDR1.

Members of the ATP-binding cassette transporter superfamily such as the P-glycoproteins (MDR) and the cystic fibrosis transmembrane conductance regulator (CFTR) share conserved sequence motifs in their nucleotide binding fold that are the major targets for CFTR mutations in patients with cystic fibrosis. Cystic fibrosis-type mutations were introduced at analogous positions into the human MDR1 gene. Heterologous expression of wild-type or mutated MDR1 revealed similar mRNA transcript levels in Chinese hamster ovary K1 recipients, but the subsequent processing was defective for all mutations that give rise to severe cystic fibrosis in the case of CFTR. Functional multidrug transporter MDR1, however, was obtained when amino acid substitutions were introduced into a less conserved position of the ATP-binding cassette transporter signature (codon 536 in MDR1). The profile of cross-resistance and chemosensitization was modulated in these codon 536 variants, which suggests that this region is involved in the drug transport function of P-glycoprotein.

Production of interleukin-1 beta and interleukin-6 in hepatoblastoma.

Thrombocytosis and fever are frequent symptoms in children with hepatoblastoma. Interleukin-6 (IL-6) has been shown to mediate thrombocytosis and an acute-phase reaction including fever. We therefore investigated samples from 14 untreated patients with hepatoblastoma for this cytokine and in addition for interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha), all of which are known to induce IL-6 production. High serum levels of IL-6 were only found in 3/14 patients; the other cytokines were not detectable. In contrast, 12/14 tumors produced substantial amounts of IL-6 in primary cell culture, while IL-1 beta was found in 3/14 supernatants; IL-1 alpha and TNF-alpha were always negative. Immunoenzymatic staining of fresh tumors revealed that IL-6 is not produced by the tumor cells, but rather by surrounding fibroblasts and endothelial cells. In tumor cells only IL-1 beta, but neither IL-1 alpha, TNF-alpha nor IL-6, could be detected. In co-culture experiments with fibroblasts and endothelial cells, addition of hepatoblastoma cells enhanced IL-6 production. Including an IL-1 receptor antagonist abolished this effect incompletely. Our results suggest that tumor cells in hepatoblastoma induce IL-6 production in surrounding fibroblasts and endothelial cells by virtue of their endogenous secretion of IL-1 beta and supposedly some other, as yet unidentified, mediator.

Expanded macrophage precursor populations in BXSB mice: possible reason for the increasing monocytosis in male mice.

The BXSB mouse spontaneously develops an autoimmune disease that resembles human systemic lupus erythematosus (SLE). During their lifetime, male BXSB mice show an increasing monocytosis in the peripheral blood as opposed to their female littermates. This monocytosis is unique among autoimmune-prone mice. To test the hypothesis that alterations at the stem cell level may be responsible for this monocytosis, myeloid bone marrow precursor cells were examined in both male and female BXSB mice from 4 to 40 weeks of age. The number of M-CSF responding stem cells (CFU-M) and the number of GM-CSF responding stem cells (CFU-GM) were higher than in all other inbred mouse strains tested. In addition, male BXSB mice developed a progressive increase of CFU-M and CFU-GM in the bone marrow during their lifetime, which paralleled the peripheral blood monocytosis. The monocytosis in male BXSB mice is the result of a further expansion of the strain-specific high number of macrophage precursors by intrinsic factors, which may be attributed to the influence of the Yaa factor. The sex-specific expanded mononuclear phagocyte system may promote the autoimmune process and may be one reason for the dramatic course of murine SLE in male BXSB mice.

Effects of human stem cell factor (c-kit ligand) on proliferation of myeloid leukemia cells: heterogeneity in response and synergy with other hematopoietic growth factors.

A novel hematopoietic growth factor, the stem cell factor (SCF), for primitive hematopoietic progenitor cells has recently been purified and its gene has been cloned. In this study we tested the mitogenic activity of recombinant human SCF on myeloid leukemia cells as well as the expression of its receptor. We have investigated the proliferation of 31 myeloid leukemia cell lines as well as fresh myeloid leukemic blasts from 17 patients in a 72-hour 3H-thymidine uptake assay in the presence of various concentrations of recombinant human (rh) SCF alone or in combination with saturating concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, M-CSF, interleukin-3 (IL-3), or erythropoietin (EPO). Only five of 31 lines, but fresh leukemic blasts from 12 of 17 patients with acute myeloid leukemia (AML), significantly responded to SCF. The responding cell lines were of the acute promyelocytic, chronic myeloid, megakaryoblastic, and erythroleukemia origin, the responding blast preparations of all French-American-British subtypes. Synergistic activities of SCF were found with G-CSF, GM-CSF, EPO, and IL-3. To determine the SCF binding sites on leukemic cells, we used 125I-radiolabeled SCF in Scatchard analysis and cross-linking studies. The leukemic cell lines responding to SCF expressed from 2,300 up to 29,000 binding sites per cell. The SCF receptor expression was downregulated in vitro by the presence of its ligand. Cross-linking studies demonstrated a 150-Kd SCF receptor on the surface of all responding myeloid leukemias. This study suggests that SCF may be an important factor for the growth of myeloid leukemia cells, either as a direct stimulus or as a synergistic factor for other cytokines. Furthermore, using polymerase chain reaction analysis of total RNA from the myeloid leukemia lines, we found expression of SCF-mRNA in 17 of 30 lines, suggesting autocrine mechanisms in the growth of a subgroup of leukemic cells by coexpression of SCF and its receptor.

Mitotic disturbance associated with mosaic aneuploidies.

The association of various unsystematic aneuploidies with premature centromere division (PCD) was observed in a patient with conspicuous clinical features and combined immunodeficiency. Trisomies and monosomies of almost all autosomes and gonosomal aberrations were found separately or in combination in a majority of the proband's lymphocytes and fibroblasts. The chromosome number varied from 44 to 50. A high proportion of the metaphases showed PCD or had the appearance of C-anaphases. These findings probably represent a new mutant affecting mitosis and causing mosaic aneuploidies.

Monoclonal antibody T-199 directed against human medulloblastoma: characterization of a new antigenic system expressed on neuroectodermal tumors and natural killer cells.

A monoclonal antibody (mAb) (T-199) of IgG1 isotype was raised against medulloblastoma by immunizations of mice with the medulloblastoma cell line TE-671. Studies of the specificity of mAb T-199 on cell lines as well as fresh frozen sections of normal and malignant tissues revealed the antigen in high amounts on the cell surface of neuroectodermally derived tumors such as medulloblastoma, neuroblastoma, retinoblastoma, and astrocytoma. Some melanomas and a subgroup of rhabdomyosarcomas also expressed the antigen. In contrast, mesenchymal tumors, osteosarcomas, and Ewing's sarcomas did not bear the T-199 antigen. Reactivity of T-199 with normal tissues has not been found with few exceptions; in certain areas of the brain, especially in the cerebellum and part of the hypothalamus, in the adrenal glands, and in the pancreatic islet cells small amounts of antigen were detectable. Natural killer cells could also be demonstrated to express the T-199 antigen similar to the NKH-1 antigen. However, despite some striking similarities, the antigens or antigen epitopes recognized by mAbs T-199 and NKH-1 are not identical. Therefore, mAb T-199 seems to detect a unique differentiation antigen on neuroectodermal tumors, coexpressed in low amounts on normal neuroectodermally derived cells and natural killer cells. The pattern of reactivity and the biochemical properties of the T-199 antigen are different from other cell surface markers for neuroectodermal cells coexpressed on natural killer cells or T-cells (HNK-1, NKH-1, or Thy-1). Biochemical analysis of the T-199 antigen showed that it is a heat-labile protein.

Inherited immunodeficiency with a defect in a major histocompatibility complex class II promoter-binding protein differs in the chromatin structure of the HLA-DRA gene.

A defect in a trans-regulatory factor which controls major histocompatibility complex class II gene expression is responsible for an inherited form of immunodeficiency with a lack of expression of human leukocyte antigen (HLA) class II antigens. We have recently described and cloned an HLA class II promoter DNA-binding protein, RF-X, present in normal B cells and absent in these class II-deficient regulatory mutants. Here we report that these in vitro results correlate with a specific change in the chromatin structure of the class II promoter: two prominent DNase I-hypersensitive sites were identified in the promoter of the HLA-DRA gene in normal B lymphocytes and found to be absent in the class II-deficient mutant cells. The same two prominent DNase I-hypersensitive sites were observed in normal fibroblastic cells induced by gamma interferon to express class II genes. Interestingly, they were also observed in the uninduced class II-negative fibroblastic cells, which have also been shown to have a normal RF-X binding pattern. We conclude that the two DNase I-hypersensitive sites in the HLA-DRA promoter reflect features in chromatin structure which correlate with the binding of the trans-acting factor RF-X and which are necessary but not sufficient for the expression of class II genes.

Congenital immunodeficiency with a regulatory defect in MHC class II gene expression lacks a specific HLA-DR promoter binding protein, RF-X.

The expression of MHC class II genes is tightly regulated. One form of congenital severe combined immunodeficiency (SCID) is characterized by a regulatory defect that precludes expression of HLA class II genes. B lymphocyte cell lines from such SCID patients provide a tool for identifying putative regulatory proteins that bind to class II gene promoters. We have identified three proteins binding to specific segments of the HLA-DRA promoter, two of which interact to form the predominant DNA-protein complex observed. One of these proteins, defined as an X box binding protein (RF-X), is specifically missing in cells from class II deficient SCID patients. We propose that the molecular defect in this congenital HLA class II regulatory deficiency is a lack of RF-X and that this factor plays an important role in the normal regulation of MHC class II gene expression.

Regulation of genes for HLA class II antigens in cell lines from patients with severe combined immunodeficiency.

HLA Class II-negative severe combined immunodeficiency (SCID) results from a congenital defect characterized by an absence of HLA Class II antigens. Patients with the disorder have no HLA-DR, DQ, or DP antigens or mRNAs in their peripheral-blood lymphocytes. The affected gene is a recessive, transacting regulatory gene that controls the expression of Class II genes. We studied the regulation of HLA Class II gene expression with the use of established Epstein-Barr virus-transformed B-cell lines and skin fibroblast lines from a group of patients with SCID. Lymphoblastoid B-cell lines from the patients contained no mRNA for HLA-DR, DQ, and DP alpha and beta polypeptides, but did express mRNA for the HLA-associated invariant chain, which is normally coregulated with HLA Class II antigens. In the B-cell line from one patient, a very low amount of DR mRNA could be detected, indicating some heterogeneity in SCID. The lymphokine gamma-interferon, a strong inducer of Class II genes in a variety of normal cells, did not restore Class II gene expression in any of the SCID B-cell lines. More important, gamma-interferon was unable to induce any Class II mRNA in fibroblast lines from patients with SCID, in contrast to the efficient induction observed in normal fibroblasts. The invariant-chain gene, however, was induced in the SCID fibroblasts, confirming a unique uncoupling in the regulation of invariant and Class II genes. Thus, the genetic defect in patients with SCID affects not only the B-cell lineage but also the inducible expression of HLA Class II genes that is normally observed in Class II-negative cells, such as fibroblasts. This unresponsiveness to gamma-interferon in vitro indicates that patients with SCID will not respond to treatment with this lymphokine. Our data also increase understanding of the normal mechanisms regulating the genes for the HLA Class II cell-surface glycoproteins.

[Myeloid antigens on human cloned natural killer cells]. / Myeloische Antigene auf menschlichen klonierten natürlichen Killerzellen.

Cloned human natural killer cells express T-cell antigens and membrane molecules which are found on myeloid cells. Both the LFA-1 antigen (CD 11a, CD 18) and the IgG Fc-receptor Fc gamma Rlow (CD 16) are functionally relevant for the activation of these cells.

Two distinct Fc receptors for IgG on human peripheral T lymphocytes.

The proportion of human peripheral T lymphocytes forming rosettes with IgG-coated ox erythrocytes (ORBC) is increased after controlled hypotonic treatment. This increment may be as high as 40% of total T cells, depending on the lymphocyte donor. Such treatment is shown not to result in selective cell loss. Induced rosetting is mediated by a receptor specific for the Fc portion of human IgG (Fc gamma R). Inhibition of induced Fc gamma R activity is equally well accomplished by monomeric and by aggregated IgG of defined size. This is in contrast to the Fc gamma R detected before hypotonic treatment, which is not significantly inhibited by monomeric IgG. Capping studies established the structural independence of these two types of Fc gamma R in the lymphocyte membrane by virtue of selective cross-linking of either receptor while leaving the respective counterpart unaffected. The biochemical basis of the hypotonic effect is not yet resolved. However, the data presented suggest that hypotonicity results in removal of Fc gamma R-bound cytophilic IgG. Operationally, we propose the term induced Fc gamma R (Fc gamma R-I) for the here-described new type of receptor with high affinity for monomeric IgG.Fc gamma R that are directly assayable without hypotonic induction and not inhibited by monomeric IgG are termed free Fc gamma R (Fc gamma R-F).

Surface antigens of human melanoma cells defined by monoclonal antibodies. I. Biochemical characterization of two antigens found on cell lines and fresh tumors of diverse tissue origin.

Monoclonal antibodies were produced against melanoma cell lines derived from patients presently disease free. Based on tissue distribution of binding, the antibodies obtained could be used to classify melanoma surface antigens into groupings similar to those obtained from studies of autologous and xenogeneic antibodies. Three antibodies (21.43, 15.75 and 15.95) reacted with the immunizing tumor but not with peripheral blood lymphocytes or a lymphoblastoid cell line derived from the tumor donor. When tested on a broader cell panel, antibody 21.43 reacted only with melanoma lines, while antibodies 15.75 and 15.95 reacted with carcinoma cell lines as well as with the majority of melanoma cell lines. Antibody 15.95 precipitated a 49000 dalton glycoprotein from both melanoma and carcinoma cells. Antibody 15.75 precipitated a 74000 dalton glycoprotein from the surface of melanoma and carcinoma cell lines which is also found on freshly isolated tumor cells from melanoma and carcinoma metastases in vivo.

Natural killer cell activity during treatment with fibroblast interferon.

Short- and long-term effects on natural cytotoxicity (NC) of i.v. administration of human fibroblast interferon (IFN--beta) was studied in patients with HBsAg positive chronic active hepatitis. Short-term kinetics demonstrated after a transient fall of NC a severalfold increase of NK activity after 24 hours. Long-term kinetics of NC revealed following features: The highest relative increase was seen during the initial phase of IFN--beta application. In all patients monitored so far the enhanced NK activity could be maintained during 2 to 4 weeks of therapy. Over a period of several weeks a gradual decrease of augmented NK activity was observed in spite of continued application of high doses of IFN--beta. These findings indicate that in-vivo administration of IFN--beta results in an augmentation of natural cytotoxicity in man. Prolonged treatment with IFN--beta seems to "exhaust" the NK cell system.

Natural and antibody-dependent cellular cytotoxicity in tumour-adherent T lymphocytes: expression of Fc receptors and of a human T-subgroup-specific antigen.

Adherence of human lymphocytes to allogeneic tumour cell monolayers was found to depend on the presence of monocytes. Adherent lymphocytes could be separated from tumour cells by treatment with lidocaine followed by nylon wool passage. Tumour-adherent cells (70% E-RFC, 45% Fc gamma-R, 23% Fc mu-R, 5% monocytes) exhibited enriched natural killer (NK) activity not only against the tumour cell line used for isolation but also against seven other targets. When T cells were isolated subsequently as E-rosettes by density gradient centrifugation through Percoll, the enrichment in cytotoxicity was even more pronounced. Tumour-adherent T cells were severalfold enriched in both NK and antibody-dependent cell-mediated cytotoxicity (ADCC) activity. However, this enrichment was not paralleled by a concomitant increase in the number of T gamma cells (tumour-adherent T cells: 17% T gamma, 40% T mu ; tumour-nonadherent T cells: 12% T gamma, 60% T mu). Marked differences could be observed by staining with a monoclonal antibody that was raised against human leukaemic T gamma cells of high NK and ADCC activity. This antibody (T 8-11) stained 60% of tumour-adherent T cells, 20% of nonadherent T cells and 29% of T-cell controls. These results indicate that the spontaneous cytotoxic activity of human T cells resides within a small population, most of which are characterized by a specific surface antigen but not by conventional Fc gamma receptors.

Efficient separation of human T lymphocytes from venous blood using PVP-coated colloidal silica particles (percoll).

In a comparative study, human peripheral T lymphocytes were separated as E rosettes by density centrifugation through various gradient media. Sheep red blood cells (SRBC) were removed by dissociation of the E rosettes at 37 degrees C with subsequent centrifugation on a similar density gradient prewarmed to 37 degrees C. In particular, gradients made of Ficoll Urovison were compared with Percoll gradients with regard to both separation steps. Using Percoll gradients, a maximal T cell recovery of 75% was obtained, whereas Ficoll separation yielded only 46%. T lymphocytes separated with Percoll exhibited equal viability compared to Ficoll isolated cells and consisted of 98% EAET-RFC. No inhibition of cellular function by Percoll treatment was detected, whereas Ficoll treatment led to an impaired mitogenic response. An inherent mitogenicity of Percoll was not observed. The method described results in considerably shortened centrifugation times due to the low viscosity of the Percoll medium and simultaneously seems to be less harmful to the rather fragile rosettes. Reproducibility was found to depend on careful control of density and osmolarity of the Percoll medium.