H. Rühl

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.

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 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.

Dual Expression of T and B Cell Markers in Human B Cell Neoplasias

Macrophage- and T-cell-depleted mononuclear cells from 36 patients with B cell lymphoma and 12 healthy individuals were investigated by indirect immunofluorescence with the monoclonal antibodies NEI-011 (7.2) and NEI-015 (10.2). The monoclonal antibody NEI-011 (7.2) recognized the Ia antigen and identified almost all B cells in the peripheral blood of healthy individuals. Furthermore, neoplastic B cells from all patients except those with plasma cell proliferation were found to react with this antibody. NEI-015 (10.2), a monoclonal antibody known to react with both T cells and B-CLL cells, did not react with normal circulating B cells. However, this antibody did identify neoplastic B cells except in cases of plasma cell proliferation and lymphoblastic lymphoma.

Adjuvant specific immunotherapy in maintenance treatment of adult acute non-lymphocytic leukemia

SummaryFrom 1976 until 1978, 136 adult patients with acute leukemia were treated in four hospitals in Berlin. A complete remission was achieved in 47 patients (35%). Twenty-six patients with non-lymphocytic acute leukemia, who had achieved a complete remission with induction chemotherapy consisting of daunorubicin (45 mg/m2/day, day 1, 2 and 3) and cytosine-arabinoside (100 mg/m2/day, continuous infusion, day 1 to day 7) were entered into a randomized trial. Thirteen patients were treated with an intermittent combination chemotherapy at 4-week intervals; the other group of patients received in addition a specific immunotherapy consisting of neuraminidase-modified allogeneic blast cells. The results revealed that the addition of this kind of immunotherapy did not increase the duration of first remission or survival.ZusammenfassungVon 1976 bis 1978 wurden 136 erwachsene Patienten mit akuter Leukämie in vier Berliner Kliniken zytostatisch behandelt. Bei 47 Patienten (35%) konnte eine komplette Remission erzielt werden. Sechsundzwanzig Patienten mit kompletter Remission und einer akuten nicht-lymphatischen Leukämie, bei denen eine Induktionstherapie mit Daunorubidomycin (45 mg/m2/Tag, Tag 1, 2 und 3) und Cytosin-Arabinosid (100 mg/m2/Tag, Dauerinfusion, Tag 1 bis Tag 7) durchgeführt wurde, wurden in einer randomisierten Studie weiter behandelt. Dreizehn Patienten erhielten in vierwochigen Intervallen eine kombinierte Chemotherapie, die andere Gruppe erhielt zusätzlich eine spezifische Immuntherapie, die in der Injektion Neuraminidase-behandelter allogenetischer Blasten bestand. Die Ergebnisse zeigen, daß durch die zusätzliche Immuntherapie in der von uns durchgeführten Form keine Verlängerung der ersten Remission oder der Überlebenszeit erreicht werden konnte.

Heterogeneity of T-cell neoplasias as defined by monoclonal antibodies

SummarySurface marker studies were carried out on neoplastic cell samples (peripheral aspirates and skin biopsies) of 302 patients with non-Hodgkin lymphomas (221 patients) and acute lymphatic leukaemias (81 patients). In 11 patients with non-Hodgkin lymphomas (5%) and eight patients with acute lymphatic leukaemia (10%), the neoplastic cells possessed phenotypic characteristics of T cells. The investigations were carried out by means of an indirect immunofluorescence technique using a panel of monoclonal antibodies (OKT 3, 4, 6, 8, 9, 10; OKM 1; HNK 1 and VIL A 1). In addition, conventional markers (SIg, E-R 4°, E-R 37°, absorbed polyclonal rabbit antithymus and anti-TDT) were used. Our results, which show a pronounced phenotypic surface marker heterogeneity between the group of T-cell neoplasias, emphasize the diagnostic value of monoclonal antisera as compared to polyclonal reagents. Eleven different surface marker profiles were observed in the 19 patients investigated.