T cells and probably B cells arise from the malignant clone in chronic myelogenous leukemia.

Bone marrow cells from a patient with Ph' positive chronic myelogenous leukemia in chronic phase were cultured for multilineage hematopoietic colonies (CFU-GEMMT), erythroid bursts, and granulocytic colonies. With CFU-GEMMT colonies, T lymphocytes were identified by reaction with monoclonal antibodies Leu-5 and OKT-3; B cells were identified by reaction with B1. All CFU-GEMMT colonies examined contained the Ph' chromosome. Recloned secondary colonies of T cells reacted with Leu-5 and OKT-3 and were Ph' positive. This demonstrates that Ph' positive T lymphocytes were generated from the pluripotential stem cell of this patient. The presence of B cells in the mixed colonies indicates that these may also be derived from the neoplastic clone.

Interleukin 9 is expressed by primary and cultured Hodgkin and Reed-Sternberg cells.

We show by mRNA hybridization analysis and immunostaining using a mouse monoclonal antibody (moAb) to recombinant human interleukin 9 (IL-9) that both primary and cultured Hodgkin and Reed-Sternberg (H-RS) cells produce IL-9 transcripts and protein and express surface binding sites for IL-9. In addition, the growth of H-RS cells obtained from the HDLM-2 line (abundantly producing IL-9 transcripts) was significantly inhibited when anti-IL-9 moAb or an IL-9 antisense oligodeoxyribonucleotide was added to cultures. Excess addition of recombinant human IL-9 relieved the effects of anti-IL-9 moAb on HDLM-2 growth. Growth of H-RS cells of the KM-H2 line, which displays only low amounts of IL-9 detectable upon hybridization of polyadenylic acid-selected RNA only, was not affected by anti-IL-9 moAb. The proliferative capacity of KM-H2 cells in soft agar and liquid suspension cultures was, however, augmented at least 3-fold when cells were exposed to recombinant human IL-9. In conclusion, our results show that IL-9 is expressed by H-RS cells and point to a possible role of this molecule as a growth factor for these cells.

Cytotoxic effector cell function at different stages of human monocyte-macrophage maturation.

Human blood-borne monocytes were cultured for up to 22 days on disposable Teflon foils. Within 8 days, these monocytes developed into mature macrophages. At various stages of differentiation, the cells were recovered from the hydrophobic membrane and were assayed for typical monocyte-macrophage enzymes and morphology, binding of monoclonal antibodies (OKM1, OKla1), Fc and transferrin receptors, phagocytic activity, lysozyme production, and ability to inhibit the growth of an allogeneic tumor target cell line (U937). A significant antitumor activity of mature macrophages was found, which developed along with the differentiation of the monocyte precursor cells. In addition, cytotoxic effector macrophages could be activated by lymphokine-rich medium and synthetic alkyl-lysophospholipids. After density gradient separation, light cells (less than 1.05 and less than 1.06 g/ml) showed enhanced cytotoxicity, whereas cells from the dense fraction (greater than 1.06 g/ml) with low base-line activity could be best activated for cytotoxicity by lymphokines. If monocyte-macrophages are involved in a natural surveillance mechanism, our results may indicate the importance of unimpaired macrophage maturation to generate effective host defense against tumor development.

Effects of human bone marrow stroma on the growth of human tumor cells.

As some tumors metastasize frequently to marrow we modified the clonogenic assay for human tumor cell growth by culturing tumor cells in the presence of human bone marrow stromal cells. In a bilayer soft agar assay, human tumor cells which had been passaged in nude mice were plated in the agar overlayer on an underlayer containing a suspension of trypsinized human bone marrow stromal cells. These marrow stromal cells stimulated the growth of tumor cells in a dose-dependent fashion, with a growth peak at a stromal cell density of 5-10 x 10(5)/ml. The maximal stimulation of tumour cell growth was 13-fold. We evaluated clonal growth of six separate tumors of five different histological types (small and large cell bronchogenic carcinoma; mammary carcinoma; malignant melanoma; pleural mesothelioma) and demonstrated that in 9 of 11 experiments tumor cell colonies formed in the absence of stromal cells, but colony growth was markedly stimulated by stromal cells in every case. Stromal stimulation persisted after irradiation of the stromal cells with 10 Gy. Growth of five fresh human tumor samples was similarly stimulated by the presence of human bone marrow stromal cells. Tumor cell colonies were characterized morphologically by Pappenheim stain and immunologically for surface antigens by peroxidase-antiperoxidase immunostaining utilizing monoclonal antibodies (carcinoembryonic antigen 26/3/13 and 26/5/1, EMA, HEA125, Sam 2 and Sam 10) which detected epithelial cell antigens. Colonies consisted of cytologically malignant cells which expressed epithelial cell antigens. Thus, the tumor cell origin of colonies from mammary carcinoma and bronchogenic small cell, large cell, and adenocarcinoma was proven. This tumor stem cell assay permits further analyses of human tumor cell biology and may be useful for testing drug sensitivity.

A prospective multicenter trial with human recombinant alpha 2c-interferon in hairy cell leukemia before and after splenectomy.

Our multicenter study on the treatment of hairy cell leukemia (HCL) started in December 1984 and the present data cover the time up to June 30, 1986. Ninety-seven patients were enrolled. For induction of response daily doses (6 micrograms) of low dose human recombinant alpha 2c-interferon (arg) was chosen. Further dose reduction (3 micrograms) was possible for patients who improved within the first 3-4 weeks. Patients with known risk factors started at lower doses (0.6 microgram daily). As infections are known to be the main cause of death in HCL, splenectomy was not mandatory before treatment. Thirty-nine patients received treatment with interferon. Nevertheless, infections remained the major cause of death in the study. The protocol did not prevent fatal infections in nine of the 34 splenectomized patients. The regimen proved safe for all but one of the nonsplenectomized patients. According to this experience, new criteria are needed for the choice of primary treatment in HCL. In our opinion splenectomy should become restricted to selected cases.

The use of a right atrial catheter in bone marrow transplantation for acute leukemia.

A right atrial catheter was implanted into 15 patients undergoing bone marrow transplantation as a treatment for acute leukemia. The catheter remained in position for 79 +/- 34 days. No catheter-related septicemia was observed. It appears that this catheter is helpful in supporting bone marrow transplant recipients.

Cyclic alternating chemotherapy of high-grade malignant non-Hodgkin lymphomas with VIM-Bleo and CHOP.

Between 1986 and 1988, 81 patients with high grade malignant non-Hodgkin lymphoma according to the Kiel classification were treated with the VIM-Bleo/CHOP-regimen: etoposide 100 mg/m2 intravenously on days 1-3, ifosfamide 1.5 g/m2 intravenously days 1-5 with mesna for prophylaxis of cystitis, methotrexate 30 mg/m2 intravenously on days 3, bleomycin 10 mg intravenously on days 8 and 15, cyclophosphamide 750 mg/m2 day 22, doxorubicin 50 mg/m2 day 22, vincristine 1.4 mg/m2 on day 22, and prednisolone 100 mg postoperatively on days 1-5 and 22-26. Cycles were repeated four times beginning on day 43. Regions with bulky disease were irradiated after chemotherapy. 36 patients (44%) had stage II, 12 (15%) stage III and 33 (41%) stage IV disease. B-symptoms were present in 49% of patients. Serum lactate dehydrogenase activity was elevated in 53%. Overall, 59 patients (73%) achieved a complete and 14 (17%) a partial remission. 8 (9%) had stable or progressive disease. After a median follow up of 30 months thus far, probability of long-term relapse free survival is 66% for patients in complete remission. Overall survival is 72% at 24 months. Toxicity from treatment was very low with leukopenia being the main side effect. Major infections were observed in only 2% of cycles with one treatment related death. VIM-Bleo/CHOP is a well tolerated regimen with remission rates in the range of other, more toxic regimens. However, cyclic alternating treatment did not improve results as compared with repeated treatment with a single standard protocol.

Role of hematopoietic growth factor combinations in experimental and clinical oncology.

Extensive in vitro studies with hematopoietic growth factors as well as numerous clinical trials with single growth factor administration provided the basis for in vivo studies with those factors in combination. Animal models and first clinical trials in humans, with the sequential application of interleukin-3 plus granulocyte-macrophage colony-stimulating factor, demonstrate that growth factor treatment in combination might be effective and could optimize hematologic responses according to specific clinical requirements. This is a brief review of some of the possible clinical applications of hemopoietic growth factors in clinical oncology with particular focus on preclinical and clinical data using combined administration. Initial experience with the combined administration of interleukin-3 and granulocyte-macrophage colony-stimulating factor in cancer patients after chemotherapy and future prospects of combinations of growth factor treatment are presented.

In vivo labelling of the spleen with a red-fluorescent cell dye.

A method for labelling mouse spleen cells in situ is described. Spleen vessels were clamped before intrasplenic injection of a red-fluorescent cell dye (PKH-26). The labelling rate was 11.8% of all cells and 13.9% of B lymphocytes 30 min after injection as determined by FACS. 3 days later, the proportions of labelled cells were reduced to 7.4% (P < 0.01) and to 10.7% (P < 0.05), respectively. Only 10% of cells detected by FACS could be detected by fluorescence microscopy. Labelled cells were not found in peripheral lymph nodes 30 min after spleen injection, but were present 3 days later (FACS: 2.8% of all cells and 5.4% of B lymphocytes, P < 0.05 each), indicating migration of stained cells. Neither adverse nor toxic effects of in situ staining were observed. Isolated stained B lymphocytes from spleens of naive mice and from lymph nodes after immunisation with insulin showed proper function when tested in an ELISA-spot assay. The labelling procedure was used to follow splenic B lymphocytes producing natural anti-insulin antibody. These cells were found to be recruited for the early immune response in lymph nodes immunised with insulin.

Demonstration of cell surface antigens and their antibodies by the peroxidase-antiperoxidase method.

The unlabeled antibody enzyme method, using the peroxidase-antiperoxidase immunocomplex for labeling, was applied to freshly prepared viable lymphocytes to detect antibodies in individuals preimmunized by pregnancies or blood transfusions. The "sandwich" incubations and washing steps were carried out with cells attached to poly(L-lysine) glass slides, in order to facilitate the handling of the samples and to save antisera and time. In comparison to the lymphocytotoxicity test, the described method is more sensitive and able to detect additional antibodies. Our findings indicate that further investigation of the use of this test for the demonstration of cell surface antigens and their antibodies appears to be worthwhile.

Immunoperoxidase slide assay (IPSA)--a new screening method for hybridoma supernatants directed against cell surface antigens compared to other binding assays.

A modified immunoperoxidase assay on microscope slides (IPSA) was adopted as a screening procedure for hybridoma supernatants with specificity for human lymphocyte subpopulations. The method proved to be suitable for testing a large number of hybridomas with a minimum of cells and reagents. In addition, the slide-technique yields considerable information about the type and morphology of target cells. This allows a first differentiation between cell types in the assayed preparation, e.g. between lymphocytes and monocytes. Compared to indirect immunofluorescence, solid-phase radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) with intact cells as antigen, IPSA turned-out to be as sensitive as RIA and superior to indirect immunofluorescence and ELISA. Nevertheless, none of the assays used detected all supernatants binding to cells in the right manner and could, thus, be considered ideal. In our hands, the combination of RIA and IPSA proved to be an excellent system for screening procedures on lymphocytes.

Lung and blood lymphocyte subsets in asbestosis and in mixed dust pneumoconiosis.

To study the local distribution of lymphocyte subsets in inorganic dust diseases, bronchoalveolar lavage (BAL) was performed in seven patients with asbestosis, and in 13 patients with mixed dust pneumoconiosis (anthracosidero-silicosis). Lymphocyte subsets in BAL and blood were determined by the monoclonal antibodies OKT3 (pan T), OKT4 (helper/inducer), OKT8 (suppressor/cytotoxic), B1 (B-cells), OKIa (HLA-DR antigens), and Leu-7 (natural killer). The BAL lymphocytes were moderately elevated to 15 +/- 8 percent (mean +/- SD) in mixed dust pneumoconiosis, and even more markedly increased to 28 +/- 21 percent in asbestosis. The OKT4/OKT8 ratio in BAL was significantly increased to 4.5 +/- 2.1 in asbestosis, and significantly reduced to 0.9 +/- 0.8 in mixed dust pneumoconiosis. In blood, the ratio of T-cell subsets remained unchanged, though total lymphocytes were decreased in asbestosis. These results suggest altered cellular immune processes in the lungs of patients with pneumoconiosis and may indicate different immunoregulatory changes depending on the nature of the inhaled dust.

Immunocytology in malignant pleural mesothelioma. Expression of tumor markers and distribution of lymphocyte subsets.

We studied the reactivity of malignant mesothelioma cells with tumor markers and the phenotypes of lymphocyte subsets in pleural effusions from 14 patients with malignant mesothelioma. For identification of cell surface antigens with monoclonal antibodies, the adhesive slide assay was used. The reaction pattern of mesothelioma cells was found to be CEA negative, Leu M1 negative, EMA positive, BMA-120 positive, My 4 positive, and BA-2 positive. The surface morphology of mesothelioma cells may be of additional help for diagnosis. By these markers, the distinction between mesotheliomas and carcinomas is facilitated. The differentiation of reactive benign mesothelial hyperplasia from malignant mesothelioma by surface marker staining is not yet possible, however. In many effusions in this study, a concomitant T-lymphocytosis was observed with a non-specific increase in the CD4/CD8 ratio, as known for other pleural diseases.

T-lymphocytosis in bronchoalveolar lavage fluid of hypersensitivity pneumonitis. Changes in profile of T-cell subsets during the course of disease.

Recently, increased proportions of OKT 4+ helper T-lymphocytes have been reported in bronchoalveolar lavage (BAL) fluid of patients with active sarcoidosis. In this study we were interested in T-cell subsets of hypersensitivity pneumonitis, a disease characterized by a similar increase in BAL T-lymphocytes as active sarcoidosis. We applied an immunoperoxidase method performed on glass slides using the monoclonal antibodies OKT 3, 4, and 8 to study T-cell subsets in blood and BAL of eight patients with hypersensitivity pneumonitis, 11 patients with active sarcoidosis, and ten control subjects. OKT 8+ suppressor cells were found to be the predominant cell type in the BAL of patients with hypersensitivity pneumonitis and recent antigen exposure. After avoidance of further antigen exposure, suppressor cells decreased and helper cells increased. The results suggest that T-lymphocytosis in BAL of hypersensitivity pneumonitis and pulmonary sarcoidosis is mediated by different immunologic mechanisms.

Alterations in immunoregulatory T-cell subsets in cigarette smokers. A phenotypic analysis of bronchoalveolar and blood lymphocytes.

Abnormalities in the proportions of immunoregulatory T-lymphocytes in bronchoalveolar lavage fluid have been reported in interstitial pulmonary diseases, yet the effect of cigarette smoking on T-cell subsets in bronchoalveolar lavage fluid from normal subjects has not been investigated. We applied an immunoperoxidase technique performed on glass slides using the monoclonal antibodies, OKT3, OKT4, OKT8, OKIa, and Leu-7 to study T-cell subsets in bronchoalveolar lavage fluid and blood of 11 normal nonsmokers and 12 smokers. In the bronchoalveolar lavage fluid of smokers, a decrease in the percentage of OKT4+ helper/inducer and an increase in OKT8+ suppressor/cytotoxic cells resulted in a markedly decreased OKT4/OKT8 ratio (0.9 +/- 0.4) compared with nonsmokers (1.9 +/- 0.8) (p less than 0.005). These abnormalities of lymphocyte subsets in bronchoalveolar lavage fluid correlated with the cumulative pack-years of smoking history but were not reflected in the peripheral blood. These results suggest that cellular immunoregulation is disturbed in the lungs of cigarette smokers. This may play a role in pulmonary defense mechanisms and tumor immunity.

Characterization of lymphoid cells by two-dimensional mini gel electrophoresis of proteins.

Cytosol proteins from normal lymphocytes, leukemic lymphocytes, and different cultured lymphoid cell lines were separated by two-dimensional mini gel electrophoresis. By staining with Coomassie blue, specific protein patterns were obtained. Very similar gel maps were produced by the cytosol proteins of chronic lymphocytic leukemia cells, hairy cells, and of in vitro grown B cells. Protein 36/6.2 (molecular weight/isoelectric point), consistently present in these cells, could not be demonstrated in normal lymphocytes. For the comparison of control Raji cells--an Epstein-Barr-Virus (EBV)-DNA carrying Burkitt's lymphoma cell line--with Raji cells induced for early antigen (EA) production, 35S-methionine-labelled total cell lysates were analyzed. In the induced cells, an additional protein (100/5.5) was found; this might be one of the immunologically defined EBV-associated antigens. These results demonstrate that two-dimensional mini gel electrophoresis can be useful for the characterization of leukemic cells in addition to the morphological, cytochemical, and surface marker analyses.

Demonstration of estrogen and progesterone receptors as well as Ki-67 and p-145 antigens in single tumor cells from blood and pleural effusions using a slide assay.

We describe a slide assay that allows the demonstration of antigens localized in the nucleus from isolated white blood cells as well as from single tumor cells derived from malignant effusions. With the antibodies Ki-67 and anti-p-145 an increased rate of nuclear and nucleolar staining resulted in cells from highly malignant lymphomas. An almost identical reaction was obtained when tumor cells from malignant effusions were tested. Cells isolated from the blood of patients with leukemic spread of lymphomas of low malignancy yielded a weak staining comparable to that of normal mesothelial cells from non-tumorous cavity fluids. The detection of estrogen and progesterone receptors (ER and PR) localized in the cell nucleus can be achieved by the same assay. The reaction is enhanced by incubation of the tumor cells for 30 min at 37 degrees C prior to fixation. Pleural effusions from 20 patients with breast cancer were tested. ER was positive in 13 and PR was positive in 12 of the 20 samples. In 5 cases there was a divergent reaction with ER and PR antibody. The hormone receptors of the primary tumor were known in 15 (ER) and 14 (PR) patients, respectively. In each cohort there was only one case with a negative reaction of the primary tumor and a positive reaction with the isolated tumor cells from the pleural effusions. These results indicate that the demonstration of hormone receptor proteins in cells from malignant effusions is possible and that there is a correlation with the status of the primary site of cancer.

Expression of the proliferation-associated Ki-67 antigen of transferrin receptors and of DNA polymerase alpha in human tumour lines: implications for in vitro chemoresistance.

To compare the time course of in vitro expression of various proliferation-associated markers including Ki-67 antigen, transferrin receptors (TfR), and DNA polymerase alpha, six human tumour cell lines of different histological origin were studied under defined conditions. Proliferation markers were demonstrated by peroxidase/anti-peroxidase staining using specific monoclonal antibodies, and their expression was compared to results obtained from [3H]-thymidine incorporation assays and cell counting. Expression of all proliferation markers began to increase during the lag phase, and occurred earlier than elevations of [3H]dT incorporation and cell numbers were recorded. Maximum expression was observed before cell growth reached plateau phase. The time courses of expression of DNA polymerase and Ki-67 were almost identical. The closest correlation of [3H]dT incorporation with time course of expression of proliferation-associated markers was observed, when intranuclear staining of DNA polymerase was analysed. TfR were expressed earlier than the polymerase and Ki-67. Since TfR were also found at remarkable levels in resting cells, they seem less proliferation-specific than Ki-67 and DNA polymerase. While in rapidly growing cell lines more than 95% of the cells expressed Ki-67, TfR, and more than 75% DNA polymerase in cell nuclei, a malignant melanoma and a pleural mesothelioma line displayed fewer than 35% of cells stained for DNA polymerase in cell nuclei during log phase. Determination of growth fractions by monoclonal antibodies may thus contribute to the prediction of chemoresistance by identifying quiescent cells that are not sensitive to S-phase-specific drugs.

Modified immunocytochemical slide technique for demonstrating surface antigens on viable cells.

Further developments of an immunoenzymatic slide technique to demonstrate cell surface antigens with monoclonal antibodies are described. In this method cells are attached to polylysine coated glass slides in order to facilitate the handling of low cell numbers and to save antibodies and time for washing the cells. The technique has been modified for the labelling of viable cells. Endogenous peroxidase is used as an additional cell marker which does not interfere with the demonstration of antigens on the cell surface by immunoperoxidase methods. Damaged cells can be identified reliably, thereby minimising interpretation errors due to non-specific antibody uptake. A double labelling technique employing peroxidase and alkaline phosphatase coupled reagents is presented. Results of this slide technique are clear cut, so that evaluation can be performed by trained technicians.

Predictive value of bronchoalveolar T cell subsets for the course of pulmonary sarcoidosis.

To study the value of knowing the proportions of bronchoalveolar T cell subsets when predicting the course of pulmonary sarcoidosis, we subjected 31 patients to clinical, physiologic, and radiographic evaluations, with controls, for at least 12 months. Initially, when all patients were untreated, BAL's were performed, and BAL lymphocyte subsets were marked by the following monoclonal antibodies: OKT3 (expressed by all T cells), OKT4 (to mark helper-inducer T cells), OKT8 (to mark suppressor-cytotoxic T cells), and OKIa (to mark Ia antigen-positive, activated T cells). A normal T4/T8 ratio was highly predictive of a favorable course: the conditions of 13 out of 15 patients with normal ratios remained stable or improved, and only 2 of these 15 patients had to be treated because of persistent symptoms. On the other hand, the conditions of 10 out of 16 patients with elevated T4/T8 ratios deteriorated during the follow-up period. The specificity of T cell subsets for predicting deterioration was improved by considering both the T4/T8 ratio and the number of Ia antigen-positive, activated T cells present. Deterioration occurred in 9 out of 11 patients with elevated T4/T8 ratios and elevated levels of activated T cells. These results suggest that the subtyping of BAL lymphocytes may be useful in determining prognosis in pulmonary sarcoidosis.