Cloning of phenotypically different human lymphocytes originating from a single stem cell.

By using hypoxanthine guanine phosphoribosyltransferase (hprt) gene alterations and chromosome aberrations as in vivo cellular markers, human T, NK, and B cells originating from a single stem cell have been successfully cloned from the peripheral blood of an atomic bomb survivor from Hiroshima. These mutant lymphocytes were selectively cloned, taking advantage of their resistance to a purine analogue, 6-thioguanine. The cloned lymphocytes possessed the same hprt gene alterations and the same chromosome aberration (20q-), but exhibited different surface or functional phenotypes and different rearrangements of TCR or Ig genes. The chromosome aberration patterns strongly suggested that the original stem cell initiated differentiation into each cell type after exposure to atomic bomb radiation. Since the person studied here was exposed to the bomb at 17 yr age, the results suggested that common stem cells exist in adults for at least T, NK, and B cells. The use of hprt gene alterations as specific cellular markers provides a novel method for identifying stem cells in the lymphocyte lineage and for studying lymphocyte differentiation in humans.

Spontaneous loss and alteration of antigen receptor expression in mature CD4+ T cells.

The TCR/CD3 complex plays a central role in antigen recognition and activation of mature T cells, and, therefore, abnormalities in the expression of the complex should induce unresponsiveness of T cells to antigen stimulus. Using flow cytometry, we detected and enumerated variant cells with loss or alteration of the surface TCR/CD3 expression among human mature CD4+ T cells. The presence of variant CD4+ T cells was demonstrated by isolating and cloning them from peripheral blood, and their abnormalities can be accounted for by alterations in TCR expression such as defects of protein expression and partial protein deletion. The variant frequency in peripheral blood increased with aging in normal donors and was highly elevated in patients with ataxia telangiectasia, an autosomal recessive inherited disease with defective DNA repair and variable T cell immunodeficiency. These findings suggest that such alterations in TCR expression are induced by somatic mutagenesis of TCR genes and can be important factors related to age-dependent and genetic disease-associated T cell dysfunction.

Evidence for increased somatic cell mutations at the glycophorin A locus in atomic bomb survivors.

A recently developed assay for somatic cell mutations was used to study survivors of the atomic bomb at Hiroshima. This assay measures the frequency of variant erythrocytes produced by erythroid precursor cells with mutations that result in a loss of gene expression at the polymorphic glycophorin A (GPA) locus. Significant linear relations between variant frequency (VF) and radiation exposure were observed for three different variant cell phenotypes. The spontaneous and induced VFs agree with previous measurements of radiation-induced mutagenesis in other systems; this evidence supports a mutational origin for variant cells characterized by a loss of GPA expression and suggests that the GPA assay system may provide a cumulative dosimeter of past radiation exposures. VFs for some survivors differ dramatically from the calculated dose response, and these deviations appear to result primarily from statistical fluctuations in the number of mutations in the stem-cell pool. These fluctuations allow one to estimate the number of long-lived hemopoietic stem cells in humans.

Gain-of-function p53 mutations enhance alteration of the T-cell receptor following X-irradiation, independently of the cell cycle and cell survival.

Missense mutations are by the far the most common types of mutations found in p53 of human tumors, suggesting that mutant p53 proteins function either by abrogating wild-type function or by gaining new oncogenic functions. To distinguish between the dominant-negative effect and gain of new function of p53 missense mutants, we measured the ability of transfected missense mutant p53s in p53-null Jurkat cells to alter T-cell receptor (TCR) surface expression. The TCR is a key signal transduction moiety common to T lymphocytes and is one of the major sites for aberrations in T-cell leukemias/lymphomas. Three p53 mutants (248trp, 249ser, and 273his) enhanced the frequency of TCR mutants after graded doses of X-radiation compared to null p53 parent- and wild-type p53-possessing normal lymphocytes; the parent Jurkat and normal lymphocyte showed no difference. These enhancements were not the results of a change in radiosensitivity or in G1 checkpoint arrest characteristics. Therefore, the creation of this mutator phenotype by missense-type p53 mutations implies that a more direct mechanism, apart from changes of cell cycle kinetics or cell death, may be responsible for the selection of certain p53 point mutations, which eventually result in the tumorigenesis of the cell.

Detection of somatic mutations at the glycophorin A locus in erythrocytes of atomic bomb survivors using a single beam flow sorter.

A modified method was developed for measuring the frequency of variant erythrocytes at the glycophorin A locus using a single beam cell sorter (SBS). Fluorescein- or phycoerythrin-labeled monoclonal antibodies specific for the M or N glycophorin A alleles were used for the SBS assay. To prevent contamination of nucleated cells in the sorting windows, the nucleated cells in the fixed erythrocyte sample were stained with propidium iodide before flow sorting. Blood samples were obtained from atomic bomb survivors who were heterozygous for the MN blood type, and the frequencies of the hemizygous and homozygous variant of the M or N glycophorin A allele were measured by the SBS. For the three types of variants, hemizygotes for M and N allele (Nø and Mø) and homozygotes for M allele (MM), the variant frequency measured by the SBS correlated well with that previously determined by a dual beam cell sorter. Variant frequencies of the Nø, Mø, and MM cell types in atomic bomb survivors determined by SBS measurements were found to increase with radiation dose (DS86, kerma) as well as with the frequency of chromosome aberrations in lymphocytes.

Detection of a circulating tumor-associated antigen with a murine monoclonal antibody, LISA 101, selected by reversed indirect enzyme-linked immunosorbent assay.

In the presence of a characterized monoclonal antibody recognizing a soluble molecule, additional monoclonal antibodies reactive with unknown antigenic determinants on the molecule can be easily selected by reversed indirect enzyme-linked immunosorbent assay. A novel murine monoclonal antibody, LISA 101, was selected by reversed indirect enzyme-linked immunosorbent assay against soluble antigens, which exist in sera and in pleural effusions derived from lung adenocarcinoma patients and which bear determinants recognized by the previously characterized murine monoclonal antibody KL-6. Antigenic determinants recognized by the LISA 101 antibody appear to be sialylated carbohydrate in nature and different from those recognized by previously reported monoclonal antibodies against sialylated carbohydrates, such as NS 19-9, FH-6, and KL-6, suggested by competitive inhibition assay and immunostaining of tissues. A circulating antigen, LISA 1-6, was detected by a bimonoclonal bideterminant assay using immobilized LISA 101 antibody and enzyme-labeled KL-6 antibody. It was found that serum LISA 1-6 levels were elevated in 63% (25 of 40) of patients with lung adenocarcinoma and in 92% (11 of 12) of patients with pancreatic carcinoma, but only in 6.5% (2 of 31) of patients with benign lung diseases and in 7.1% (1 of 14) of patients with pancreatitis. The present observations indicate that the LISA 1-6 antigen may serve as a new tumor marker for adenocarcinomas of the lung and the pancreas. Additionally, the reversed indirect enzyme-linked immunosorbent assay may be a widely applicable method for selecting new monoclonal antibodies against as yet unknown antigenic determinants on soluble molecules.

Monoclonal antibodies to human squamous cell carcinoma of the lung and their application to tumor diagnosis.

Three immunoglobulin G1 monoclonal antibodies, LuCa2, LuCa3, and LuCa4, were produced by fusing murine myeloma NS1 cells with splenocytes obtained from a BALB/c mouse immunized with SK-MES1 cells derived from human squamous cell carcinoma of the lung. These three monoclonal antibodies were shown to recognize different protein antigens on SK-MES1 cells by indirect immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. While the pattern of cell line distribution of antigens recognized by these antibodies was not tumor type specific, their reactivity with tissue and pleural effusion was much more informative than with cell lines. The presence of target antigens in vivo was analyzed by immunoperoxidase staining of frozen tissue sections and immunofluorescence staining of tumor cells in pleural effusions. LuCa2 antibody was reactive with lung squamous carcinoma and adenocarcinoma tumor tissues and pleural effusions, but only infrequently with those of small cell carcinoma. This antibody was also reactive with many tumor tissues from other organs as well as with various normal tissues, including alveoli and bronchus. LuCa3 and LuCa4 antibodies reacted with lung squamous carcinoma in tissues and pleural effusions, but not with lung adenocarcinoma nor with small cell carcinoma. These two antibodies reacted only weakly with normal squamous tissues of the esophagus, skin, and cervix uteri, but not with various other normal tissues. Moreover, LuCa3 had weak reactivity with squamous cell carcinoma tissue of tongue and esophagus, whereas LuCa4 had no reactivity with nonpulmonary tumor tissues. LuCa3 and LuCa4 antibodies should be of clinical interest, because our data suggest that these antibodies may be potentially useful for the diagnosis of the histological type of lung tumor cells in both cancer tissue and pleural effusions.

Continued expression of a tissue specific activated oncogene in the early steps of radiation-induced human thyroid carcinogenesis.

Ionizing radiation is a well-known risk factor of cancer development, but the mechanism of radiation induced carcinogenesis is not clear. Chromosomal rearrangements induced by radiation most likely are one of the principal genetic alterations resulting in malignant transformation. The chimeric BCR-ABL associated with chronic myelogenous leukemia (CML) and H4-RET oncogenes associated with thyroid papillary carcinoma are the result of a translocation and inversion, respectively. In vitro studies showed these genes were induced by high-doses of X-irradiation in cell lines. Studies also show that therapeutic external X-ray doses as high as 60 Gy for treatment of various childhood cancers including Hodgkin's disease significantly increase the risk of thyroid cancer. Therefore, we examined the induction and persistence of these chimeric genes in human thyroid tissues transplanted in scid mice after 50 Gy exposure as a function of time for 2 months to elucidate the early events of thyroid carcinogenesis. The H4-RET genes were detected on day 2 and throughout the 2 month period. On the other hand, BCR-ABL genes were detected on day 2 and were undetectable subsequently. These results suggest that ionizing radiation causes various oncogene activations, but cells with only specific gene alteration uniquely associated with thyroid carcinogenesis are selectively retained demonstrating one of the early events in the beginnings of radiation carcinogenesis in human thyroid tissues.

Preferential induction of RET/PTC1 rearrangement by X-ray irradiation.

Ionizing radiation is a well known risk factor of thyroid cancer development, but the mechanism of radiation induced carcinogenesis is not clear. The RET/PTC oncogene, an activated form of the RET proto-oncogene, is frequently observed in papillary thyroid carcinoma (PTC); RET/PTC1, -2 and -3 are known to be the three major forms. High frequencies of RET/PTC rearrangements have been observed in radiation-associated PTC, such as those appearing post-Chernobyl or post-radiotherapy, but the rearrangement types differ between these two populations. We investigated whether a specific type of RET/PTC rearrangement was induced by X-rays in vivo and in vitro. In human normal thyroid tissues transplanted in scid mice, the RET/PTC1 rearrangement was predominantly detected throughout the observation period (up to 60 days) after X-ray exposure of 50 Gy. On the other hand, RET/PTC3 was detected only 7 days after X-irradiation, and no transcript of RET/PTC2 was detected. These results are supported by the results of an in vitro study. The RET/PTC1 rearrangement was preferentially induced in a dose-dependent manner by X-rays within a high dose range (10, 50 and 100 Gy) in four cell lines. On the other hand, RET/PTC3 was induced at a much lower frequency, and no induction of RET/PTC2 was observed. These results suggest that the preferential induction of the RET/PTC1 rearrangement may play an important role in the early steps of thyroid carcinogenesis induced by acute X-irradiation.

A lectinlike receptor on murine macrophage cell line cells, Mm1: involvement of sialic acid-binding sites in opsonin-independent phagocytosis for xenogeneic red cells.

The recognition mechanism of xenogenic red cells by mouse macrophages was studied by using established cell lines. Approximately 30% of cell line cells Mm1 which lack la antigen, as well as of thioglycollate-induced peritoneal macrophages from SL/Am mice (TGC-M phi) could ingest unopsonized quail red cells (QRC). In contrast, an undifferentiated type of cell line, M1-, and another type of macrophage cell line, Mk1-C, possessing accessory cell activity in association with the expression of la antigen, had no phagocytic activity for QRC. Approximately 80% of Mm1 cells, as well as TGC-M phi formed rosettes with QRC, whereas M1- and Mk1-C cells did not; indicating that specific binding sites for QRC are expressed on a large portion of Mm1 and TGC-M phi but not on M1- and Mk1-C cells. No requirement of divalent cation (Mg++, Ca++) and metabolic energy was observed for rosette formation between Mm1 cells and QRC. Protease treatment of Mm1 cells eliminated the rosetting activity, whereas periodate oxidation of glycosidase treatment slightly enhanced this activity, suggesting the involvement of surface protein in binding sites of Mm1 cells. In contrast to these findings on Mm1 cells, binding components of QRC were sensitive to periodate oxidation or neuraminidase treatment but resistant to protease, suggesting that the terminal sialic acid residues of carbohydrate of QRC are recognized by Mm1 cells. Furthermore, N-acetylneuraminic acid (NeuNAc) inhibited the rosette formation and promoted the dissociation of rosettes already formed. N-Acetylneuramin lactose (Neu-NAc-Lact) was more efficient in rosette inhibition than NeuNAc. These sugars also blocked the phagocytosis of QRC by Mm1 cells but had no effect on either Fc-mediated phagocytosis or latex ingestion. These results suggest that phagocytosis of QRC by murine macrophages is mediated by protease-sensitive binding sites recognizing terminal sialic acid residues of QRC in conjunction with additional carbohydrates.

Cryopreservation of human lymphocytes for assessment of lymphocyte subsets and natural killer cytotoxicity.

Cryopreservation of lymphocytes has become increasingly important, especially when the cells are to be used in retrospective studies of selected and dwindling populations, such as A-bomb survivors. This report describes an efficient method for cryopreservation of human lymphocytes which does not significantly alter various immunological characteristics of these cells. The proportions of Leu-1+ cells (T cells), Leu-2a+ cells (suppressor-cytotoxic T cells), Leu-3a+ cells (helper-inducer T cells), HLA-DR+ cells, Mo2+ cells (monocytes), B1+ cells (B cells), and Leu-7+ cells (natural killer (NK) cells), as determined by monoclonal antibodies, were found to be stable following cryopreservation. NK cell activity against K-562 target cells showed a 40-60% decrease immediately after thawing, but recovered to approximate pre-freezing levels after preincubation for 18 h. Neither lymphocyte subsets nor cell viability significantly changed following preincubation after cryopreservation. However, the ratio of cells binding to K-562 cells increased after this preincubation and may account for the observed recovery of NK cell activity. NK cell activity remained relatively stable up to 14 months of storage which confirms that freezing damage depends on the freezing process rather than on the duration of cryopreservation.

Isolation and characterization of human peripheral blood CD4+ T cell clones expressing gamma delta T cell receptors.

Rare T cell clones bearing both CD4 and gamma delta T cell receptors (TcR gamma delta) were obtained from human peripheral blood by cell sorting using anti-CD4 and anti-TCR delta 1 antibodies. All the clones established were reactive with anti-TcR gamma delta 1 antibody, whereas only about 20% of the clones showed reactivity with anti-delta TCS1 antibody. Unlike most CD4+ T cells bearing TcR alpha beta, all the clones tested showed lectin-dependent and anti-CD3 antibody-redirected cytolytic activity. About 60% of the clones exhibited natural killer cell-like activity. Immunoprecipitation analysis of TcR gamma delta showed that each clone expressed either a disulfide-linked or non-disulfide-linked heterodimer consisting of 37-44-kDa TcR gamma and TcR delta chains.

Lifespan of human memory T-cells in the absence of T-cell receptor expression.

To evaluate the intrinsic lifespan of human memory T-cells in the absence of T-cell receptor signaling, we used radiation-induced mutant CD4+ T-cells lacking surface expression of TCR/CD3 complex as an in vivo cell marker. We analyzed the long-term kinetics of TCR/CD3 - mutant T-cells among CD4+ CD45RA+ naive and CD4+ CD45RA- memory T-cell fractions in peripheral blood of gynecological cancer patients receiving radiotherapy. Both the proportion and number of these mutant T-cells decayed exponentially with time following radiotherapy. The estimated half-life of mutant memory T-cells was 2 to 3 years and did not differ from that of mutant naive T-cells. These results indicate that the lifespan of mature CD4+ T-cells is limited regardless of their memory or naive phenotype in the absence of TCR/CD3 expression. This finding may suggest that continued T-cell receptor signaling is required for lifetime maintenance of human memory T-cells.

The usefulness of severe combined immunodeficiency (SCID) mice to study human carcinogenesis.

In the present study, we engrafted normal colonic epithelial and histologically diagnosed colonic adenomas from a familial adenomatous polyposis (FAP) patient into severe combined immunodeficient (SCID) mice and subsequently examined them histologically and molecular biologically. Successful engraftment and metastasis was observed. The facts that human normal colonic epithelium and adenomatous polyps can take in SCID mice indicates the possibility that this human SCID mouse system will be useful for investigating the dynamics of human carcinogenesis in various tissues.

Monitoring exposure to atomic bomb radiation by somatic mutation.

Atomic bomb survivors are a population suitable for studying the relationship between somatic mutation and cancer risk because their exposure doses are relatively well known and their dose responses in terms of cancer risk have also been thoroughly studied. An analysis has been made of erythrocyte glycophorin A (GPA) gene mutations in 1,226 atomic bomb survivors in Hiroshima and Nagasaki. The GPA mutation frequency (Mf) increased slightly but significantly with age at the time of measurement and with the number of cigarettes smoked. After adjustment for the effect of smoking, the Mf was significantly higher in males than in females and higher in Hiroshima than in Nagasaki. All of these characteristics of the background GPA Mf were in accord with those of solid tumor incidence obtained from an earlier epidemiological study of A-bomb survivors. Analysis of the dose effect on Mf revealed the doubling dose to be about 1.20 Sv and the minimum dose for detection of a significant increase to be about 0.24 Sv. No significant dose effect for difference in sex, city, or age at the time of bombing was observed. Interestingly, the doubling dose for the GPA Mf approximated that for solid cancer incidence (1.59 Sv). And the minimum dose for detection was not inconsistent with the data for solid cancer incidence. The dose effect was significantly higher in those diagnosed with cancer before or after measurement than in those without a history of cancer. These findings are consistent with the hypothesis that somatic mutations are the main cause of excess cancer risk from radiation exposure.

Growth suppressive efficacy of human lak cells against human lung-cancer implanted into scid mice.

The purpose of our study was to determine the efficacy of immunotherapy using human lymphokine activated killer (LAK) cells against a human-lung squamous-cell carcinoma cell line (RERF-LC-AI) implanted into severe combined immunodeficient (SCID) mice. A statistically significant growth suppressive effect on RERF-LC-AI implanted into SCID mice was observed when human LAK cells were administered into the caudal vein of the mice treated with a continuous supply (initiated prior to LAK cells injection) of rIL-2. The human LAK cells stained with PKH 2, a fluorescent dye, for later detection using flow cytometry were administered into the caudal vein of RERF-LC-AI bearing SCID mice; the cells persisted for 7 days in the implanted lung cancer tissue and in the mouse peripheral blood, but for 5 days in the mouse spleen. The number of infiltrated human LAK cells in each tissue increased dose-dependently with the number of injected cells. The results indicate that the antitumor effect most likely occurred during the early implantation period of the human LAK cells. These results demonstrate the applicability of this model to the in vivo study of human lung cancer therapy.

Scid mice model for the in-vivo study of human oncotherapy - studies on the growth and metastasis of human lung-cancer.

For the study of the growth and metastasis of human lung cancer, we established a severe combined immunodeficient (SCID) mouse model for engraftment of intact human lung-cancer tissue dissected from patient specimens. Small fragments of human lung-cancer tissues (14 cases) obtained from surgery or autopsy were implanted into the mammary fat pads of SCID mice. Seven of the fourteen cases (50%) showed an evident enlargement of the implanted lung-cancer tissue, the histopathology of which was almost identical to that of the original cancer tissues for as long as 2 months following implantation. There was slight correlation between the implantation success rate and the clinical stage of the patient at implantation. A second implantation of cancer tissues on four of these cases was successful. In contrast, no significant enlargement of the implanted tissue was observed in the cases of normal human peripheral-lung tissues (five cases), but a bronchial epidermal feature was observed in all of them. Matrigel (Collaborative Research, Bedford, MA) coating of the tissues significantly increased the growth rate of lung-cancer implants, and a high correlation (R=O.806) between the size of the implanted human lung-cancer tissues and carcinoembryonic antigen levels in the SCID mice was seen. Additionally, human lung-cancer cell lines subcutaneously injected into the backs of mice showed more metastatic lesions in the lungs and lymph nodes of SCID mice than in nude mice. Also, fresh human lung cancer metastasized to the lymph nodes and lungs of SCID mice. The results demonstrate the utility of SCID mice as recipients of human lung-cancer tissue and the applicability of this model to the in vivo study of mechanisms of human lung-cancer growth and metastasis.

A circulating heat-resistant mucin-like antigen in patients with lung-cancer detected by a new murine monoclonal-antibody.

We discovered a circulating mucin-like antigen designated as CAM-14 detected by a new murine monoclonal antibody KL-14 (IgM). We found different heat resistant properties between serum CAM- 14 from normal individuals and from lung cancer patients. Heat treatment had less effect on the levels of CAM-14 in sera from lung cancer patients, whereas CAM-14 levels in sera from normal individuals were markedly decreased after heat treatment at tempratures > 65-degrees-C. As a serum tumor marker, CAM- 14 had only very low levels of false-positive values with a high specificity and effectively increased the positive rate for lung cancer patients when used together with carcinoembryonic antigen.

New serum indicator of interstitial pneumonitis activity. Sialylated carbohydrate antigen KL-6.

Serum levels of a high molecular weight circulating antigen KL-6, detected by means of a sandwich assay using a monoclonal antibody KL-6 against a sialylated carbohydrate antigen, were evaluated for usefulness in monitoring the activity of interstitial pneumonitis. Abnormally high levels of KL-6 antigen were observed in the sera of 34 (58 percent) of 59 patients with interstitial pneumonitis. There was no significant correlation between serum values of KL-6 antigen and LDH activity. There was a positive correlation between KL-6 antigen levels and the degree of clinical disease activity as measured by 67Ga-citrate scintigram and the clinical course. Though this is a preliminary study, these observations suggest that the serum level of KL-6 antigen may be a useful indicator of disease activity in patients with interstitial pneumonitis. It does not appear to be useful, however, in the differential diagnosis of interstitial pneumonitis from malignant and nonmalignant diseases.

Direct evaluation of radiation damage in human hematopoietic progenitor cells in vivo.

We have developed techniques by which normal functional elements of human bone marrow can be implanted into immunodeficient C.B-17 scid/scid (SCID) mice. Afterward, long-term multilineage human hematopoiesis is sustained in vivo. We evaluated the effect of irradiation on the function of human bone marrow with this in vivo model. After whole-body X irradiation of the engrafted animals, it was determined that the D0 value of human committed progenitor cells within the human marrow was 1.00 +/- 0.09 (SEM) Gy for granulocyte-macrophage colony-forming units (CFU-GM) and 0.74 +/- 0.12 Gy for erythroid burst-forming units (BFU-E). The effects of irradiation on the hematopoietic elements were reduced when the radioprotective agent WR-2721 was administered prior to irradiation. After low-dose irradiation, recovery of human myelopoiesis was accelerated by treatment with human granulocyte colony-stimulating factor (G-CSF). This small animal model may prove amenable for the analysis of the risk of the exposure of humans to radiation as well as for the development of new modalities for the prevention and treatment of radiation-induced hematopoietic damage.