G. Sieber

Chronic myelocytic leukemia as a second neoplasia in the course of chronic lymphocytic leukemia—Case report and review of the literature☆

A patient with chronic lymphocytic leukemia developed Ph1-positive chronic myelocytic leukemia after a 6-yr course of CLL. Chemotherapy for CLL consisted of chlorambucil and steroids, later vincristine and bleomycin; after resistance to these agents, cyclophosphamide, vincristine and prednisolone were applied. When CML was diagnosed, we found two morphologically distinct populations of malignant cells in the bone marrow; the Ph1-chromosome was identified, and immunological surface marker studies also demonstrated two distinct malignant cell populations. Up to now, only five cases of CML have been reported following CLL and one case accompanying it. Three patients were treated with cytostatic drugs, one patient by total body irradiation and two patients received no therapy. At present, it is not clear whether the development of CML during CLL represents a therapy-induced complication or an increased susceptibility to second malignancies due to the leukemic process itself or possibly to immunological deficiencies in CLL. Since two patients received no treatment for CLL, previous therapy does not seem to be a prerequisite for the development of CML.

Immunosuppressive effects of recombinant interferon‐α during long‐term treatment of cancer patients

Interferons (IFN) are known to modulate immune responses in an either stimulatory or inhibitory manner. Most of the knowledge about immunomodulatory activities of IFN comes from investigations of IFN effects on cells in vitro. This study examines the influence which long‐term treatment with recombinant interferon‐α2 exerts on immune functions in cancer patients. Serial in vitro immune function studies of peripheral blood mononuclear cells were done to determine parameters of B‐cell and T‐cell functions as well as natural killer (NK)‐cells activity. The authors detected profound suppression of in vitro immunoglobulin synthesis and lymphocyte proliferation as well as depression of NK‐cell activity during IFN treatment. All suppressed immune functions normalized on discontinuation of IFN therapy. The authors conclude from these observations that, apart from their beneficial effects, IFN produce substantial immunosuppression.

Characteristics of immunoglobulin secretion in man evaluated by a reverse hemolytic plaque assay.

ZusammenfassungMit Hilfe eines Plaque Assays wurde die Kinetik der durch Pokeweed Mitogen induzierten Transformation menschlicher B-Lymphozyten in Immunglobulin-sezernierende Zellen untersucht. Als Indikatorzellen dienten Schafserythrozyten, an die Protein A gekoppelt war. Die maximale Stimulation wurde in allen Experimenten nach einer Kulturdauer von 6–8 Tagen erreicht. Nach Optimierung der Versuchsbedingungen erwies sich dieser Plaque Assay als ausgezeichnete Methode zur quantitativen Bestimmung der Anzahl Ig-sezernierender Zellen vitro; er war außerdem gut geeignet, die B-Zellfunktion eines Individuums über mehrere Monate zu verfolgen.Die Ergebnisse haben gezeigt, daß die durch Pokeweed Mitogen bewirkte B-Zellaktivierung an das Vorhandensein von Helfer-T-Zellen gebunden war. Außerdem konnte mit anderen, typischen T-Zellmitogenen, ebenfalls eine polyklonale B-Zellaktivierung bewirkt werden. — In weiteren Experimenten wurde gezeigt, daß die Ko-Kultivierung von Lymphozyten zweier nicht HLA-identischer Spender nicht zu einer B-Zellaktivierung führt; nach Stimulierung mit Pokeweed Mitogen wurde auch in diesen Kulturen eine Aktivierung gefunden, die dem erwarteten Ausmaß entsprach. Diese Befunde lassen den Schluß zu, daß mit dieser Technik wertvolle Ergebnisse in der experimentellen und klinischen Immunologie gewonnen werden können.SummaryThe kinetics of Pokeweed mitogen-induced transformation of human B-lymphocytes into immunoglobulin-secreting cells were examined in a reverse hemolytic plaque assay using protein A-coated sheep red blood cells as indicator cells. Peak responses occurred consistently after 6–8 days of incubation. After determination of the optimal experimental conditions the RHPA was found to be a reliable tool to estimate ISC in vitro; the technique was also found to be applicable for experiments surveying the B-cell response of an individual over a period of months.The PWM-induced transformation of B cells was absolutely T-cell-dependent. Other substances known as typical T-cell mitogens were also tested for polyclonal B-cell activation and some of them showed significant responses. Further experiments have shown that co-cultivation of non-HLA identical cells did not increase the number of plaques in unstimulated cultures whereas the addition of PWM leads to the generation of ISC within the expected range. These findings open a wide field of application of the RHPA in experimental and clinical immunology.

Abnormalities of B-Cell activation and immunoregulation in splenectonlized patients

Using a reverse hemolytic plaque assay as the effector system, we studied B-lymphocyte function in 12 patients after posttraumatic splenectomy, as well as in 25 normal individuals. The time interval between the splenectomy and the immunological studies varied between 2 days and 7 years. Compared to normal individuals, the splenectomized patients had markedly elevated numbers of spontaneous immunoglobulin-secreting cells (ISC) and severely decreased responses to the polyclonal activator pokeweed mitogen. A tendency towards normalization of these abnormalities, especially the high spontaneous ISC levels, could be observed during the time interval extending up to 7 years after splenectomy. In order to characterize the mechanism responsible for the altered immune response in splenectomized patients, co-culture experiments with unseparated and separated lymphocytes were performed. These revealed an impaired T-helper cell capacity as well as an intrinsic B-cell defect. Marker analyses with monoclonal antibodies revealed normal proportions with the exception of OKT 4 positive and B 1 positive cells that identify T-helper/inducer and peripheral B-cells respectively. We conclude that immune dysfunction in peripheral blood lymphocytes of splenectomized patients involves mainly the B-cell as well as the T-helper/inducer-cell population.

Expanded Tγ cell populations with the morphology of large granular lymphocytes — I. Immunological, clinical and morphological characterization☆

Lymphocytes from two patients with T gamma cell proliferations displaying the morphology of large granular lymphocytes (LGL) were characterized in terms of cell marker phenotyping and immunologic functions. In both patients, the lymphocytes were positive for E-R, HuTLA, OKT5, OKT8, OKT11, OKM1, VEP13, Leu1, Leu2a, Lyt2 and Lyt3 and were negative for Tmu, Tar, SIg, BA1, BA2, EM-R, C3d-R, C3b-R, OKT6, OKT9, Leu3a, OKIa1 and TdT. In addition, investigations for T411, T811 and M522 in patient 1 yielded positive results. There were differences in the phenotype of the two patients with regard to the reactions with OKT3, OKT10 and VEP10. While, in patient 1, OKT3 was very pronounced and OKT10 and VEP10 were completely negative, OKT10 and VEP10 were very pronounced in patient 2, whereas OKT3 was positive only in a very small percentage of cells. Though the lymphocytes in both patients were potent effectors of NK and K functions (patient 2 more strongly than patient 1) and a noticeably reduced mitogen response was shown to PHA, Con A and zinc, patient 1 showed a distinct suppression of allogenic and autologous B cell response to transformation into ISC, coinciding with the clinical observation of a hypogammaglobulinemia; neither B cell suppression nor dysgammaglobulinemia was seen in patient 2. The results are discussed with regard to other comparable T gamma proliferations reported in the literature.

Evaluation of the circulating and splenic lymphocyte subpopulations in patients with non-Hodgkin lymphomas and Hodgkin's disease using monoclonal antibodies.

SummaryThe number of B lymphocytes, T lymphocytes and their helper/inducer, cytotoxic/suppressor and NK/K subpopulations was measured in peripheral blood and spleen cell suspensions from patients with Hodgkins disease (HD) in the active stage of the disease and in remission status, as well as in Non-Hodgkin lymphomas (NHL) in active stage of the disease. B lymphocytes were determined by direct immunofluorescence and T lymphocytes with the E rosette technique. Helper/inducer, cytotoxic/suppressor, and NK/K T lymphocytes were determined by indirect immunofluorescence with the monoclonal antibodies OKT4, OKT8 and Leu7 (HNK1). In the same way, Lyt3 was used for determination of the total T lymphocytes. Whereas in peripheral blood of the NHL group an increase of B lymphocytes and a slight reduction of T lymphocytes could be observed, with normal distribution of the subpopulations, in patients with active HD as well as in those in remission, a marked absolute and relative decrease of T helper/inducer cells was found with normal cytotoxic/suppressor and NK/K proportion. In contrast to this, a significant increase of helper/inducer T lymphocytes with decreased cytotoxic/suppressor T proportion was found in spleen cell suspensions of patients with HD.

Lymphocyte Subsets in the Peripheral Blood of Patients with Multiple Myeloma and Benign Monoclonal Gammopathy

SummaryA panel of monoclonal antibodies that identify various antigens present on T and B cells was used to characterize circulating blood lymphocyte subsets in multiple myeloma [(MM) - 13 patients] and benign monoclonal gammopathy [(BMG) - 5 patients]. In MM and BMG an increase in B cell proportions (BA 1 positive cells) was observed, whereas T cells (Lyt 3 positive cells) were reduced compared to normal controls. However, with respect to the T cell subset distribution, a marked diversity between MM and BMG was noted. This may help to differentiate BMG from MM. In MM a relative decrease in inducer/helper T cells (OKT 4 positive cells) and increase in suppressor/cytotoxic T cells (OKT 8 positive cells) as well as in NK/K T cells (Leu 7 positive cells) was observed. On the other hand, in BMG the relative T cell subset distribution was comparable to those seen in normal subjects.

Impaired B lymphocyte reactivity in patients after radiotherapy

The effect of therapeutic irradiation upon B lymphocyte function was investigated in patients with various malignancies. The test system used was a reverse hemolytic plaque assay, which made it possible to study the activation and differentiation of B lymphocytes into immunoglobulin-secreting cells (ISC). Peripheral blood lymphocytes from normal individuals and patients before and after radiotherapy were stimulated in vitro with the polyclonal B cell activator pokeweed mitogen, and the number of ISC was estimated. B cell reactivity was markedly reduced in those patients who had received irradiation within the last six months. In patients in whom radiotherapy had been terminated more than 12 months before the lymphocytes were tested, B cell reactivity was comparable to that of patients prior to radiotherapy. By means of marker analyses, there was a reduction of B lymphocytes and T lymphocytes in the peripheral blood with a preponderance of T helper cells. Several mechanisms--e.g., reduced or defective B cell differentiation, altered regulatory T-helper or suppressor cell function or activation of suppressive monocytes--could be responsible for impaired B cell reactivity after radiotherapy.

Impaired B-lymphocyte reactivity in patients with Hodgkin's disease and non-Hodgkin-lymphomas.

SummaryThe generation of immunoglobulin-secreting cells upon stimulation with pokeweed mitogen was studied in patients with Hodgkins Disease (HD) and non-Hodgkin lymphomas (NHL) using a reverse hemolytic plaque assay. The experiments revealed that the majority of the patients in both groups had greatly diminished responses of their peripheral B-lymphocytes. Whereas in NHL patients an intrinsic B-cell defect is the most likely explanation for the impaired B-cell reactivity, the situation in HD appears to be more complex. In some patients with an active stage of the disease, suppressor cells were found to cause the unresponsiveness: in other patients, an intrinsic B-cell defect as well as a defect of the T helper cells were responsible for the diminished responses. The results further suggest that the immune defect in patients with HD is reversible and can be corrected by therapy.

B-cell function in patients with chronic lymphocytic leukemia

SummaryUsing a reverse hemolytic protein A plaque assay, spontaneous and pokeweed mitogen (PWM)-induced immunoglobulin (Ig) secretion was determined in peripheral blood from 22 patients with B1-chronic lymphocytic leukemia (CLL), one patient with B2-CLL, and one patient with suppressor T-CLL. Diagnoses were established by cytological and histological criteria as well as several marker analyses. Lymphocytes from B1- and B2-CLL patients were unable to secrete Ig either spontaneously or after PWM stimulation. In T-CLL lymphocytes, spontaneous Ig secretion was suppressed very probably by the OKT-8-positive leukemic population, since, after cultivation with PWM, a normal Ig secretion could be demonstrated which was paralleled by a decrease in the OKT-8-positive cells. Cocultivation experiments with freshly isolated, unseparated lymphocytes from normal subjects and lymphocytes from patients were of no informational value, since isolated normal B-cells alone already showed a high rate of Ig secretion. However, coculture experiments with separated subpopulations after PWM stimulation revealed an intrinsic B-cell defect in lymphocytes from B1-CLL patients, whereas their T-lymphocytes were found to be normal helper cells.ZusammenfassungMittels eines indirekten Protein A Plaque Assays wurden im peripheren Blut von 22 Patienten mit B1-CLL, einem Patienten mit B2-CLL und einem Patienten mit einer Suppressor-T-CLL die Lymphocyten bestimmt, die spontan bzw. nach Stimulation mit Pokeweed Mitogen Immunglobuline synthetisieren. Die Diagnosen wurden mit Hilfe der Cytologie und der Histologie sowie anhand von Markeruntersuchungen gestellt.Lymphocyten von Patienten mit B1- und B2-CLL waren weder spontan noch nach Pokeweed Mitogen-Stimulation in der Lage, Immunglobuline zu sezernieren. Im Falle der T-CLL war die Spontansekretion höchstwahrscheinlich durch die OKT 8 positive Leukämiepopulation supprimiert, da nach Kultivierung mit Pokeweed Mitogen eine normale Immunglobulin-Sekretion — verbunden mit einer Abnahme der OKT 8 positiven Zellen — festzustellen war. Ko-kulturexperimente mit frisch isolierten, ungetrennten Lymphocyten von Normalpersonen und Lymphocyten von Patienten waren nicht aussagekräftig, da isolierte normale B-Zellen allein schon eine sehr hohe Immunglobulinsekretionsrate aufwiesen. Dagegen erbrachten Ko-kulturexperimente mit Subpopulationen von B1-CLL Patienten den Beweis für einen wirklichen B-Zelldefekt, während die entsprechenden T-Lymphocyten als normale Helferzellen fungieren konnten.