J. Wessels

Relationship between internalization and cytotoxicity of ricin A-chain immunotoxins

Immunotoxins (ITs) appear to vary considerably in their killing efficiency towards antigen positive cells. In order to unravel the mechanisms underlying these differences, the parameters responsible for these variations were studied. The efficacy of the monoclonal antibodies (MoAbs) WT32 (CD3), OKT4 (CD4), T101 (CD5), WT1 (CD7) and WT82 (CD8) conjugated to ricin A‐chain was expressed by the extent of protein synthesis inhibition of four leukaemic T cell lines (CEM, GH1, Jurkat and HPB‐ALL). Large differences in cytotoxicity were observed. Efficient inhibition of protein synthesis was seen with anti‐CD3 IT, anti‐CD5 IT and anti‐CD7 IT. In these cases the cytotoxicity was related to the antigen density on the target cell membrane. Anti‐CD4 IT inhibited poorly and anti‐CD8 IT was ineffective, even on cell lines with a high expression of the corresponding antigen. When antigen density and cytotoxicity were plotted for all CD antigens, no correlation could be found. Subsequently, internalization was studied with 125Iodine‐labelled antibodies. Anti‐CD7 showed the fastest internalization rate, followed by anti‐CD3 and anti‐CD5. Anti‐CD4 and anti‐CD8 antibodies were respectively moderately and hardly internalized. When the absolute amount of internalized MoAb was calculated, a highly significant correlation with cytotoxicity was found. We conclude that the degree of antigen expression is not so important as the absolute amount of antibody internalized in predicting the efficacy of ITs.

In vitro response of blasts to IL-3, GM-CSF, and G-CSF is different for individual AML patients: factors that stimulate leukemic clonogenic cells also enhance Ara-C cytotoxicity.

SummaryIn vivo, growth factors are currently investigated for their capacity to trigger leukemic stem cells into cycle and thus overcome kinetic drug resistance. In this study, the susceptibility of leukemic clonogenic cells to individual growth factors was related to cytosine-arabinoside (Ara-C) sensitivity. The effects of interleukin-3 (IL-3), granulocyte-macrophage colonystimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and combinations of these recombinant hematopoietic factors were tested on blast cells of nine acute myeloid leukemia (AML) patients. Growth factor responses were assessed in semi-solid clonogenic assay and in a 10-day liquid culture followed by clonogenic assay. Heterogeneity in growth factor response was observed in both test systems, resulting in a variable pattern for individual leukemias. In the majority of cases (six of nine) the response patterns in the semi-solid and liquid cultures were divergent. To test the Ara-C sensitivity, leukemic blasts were exposed in liquid to various concentrations of Ara-C in the absence and presence of preselected growth factors. After 10 days, the number of surviving leukemic colony-forming cells (CFU-L) was assessed. Exposure to Ara-C in the presence of optimal stimulatory factor(s) resulted in a 3- to 1000-fold increase of the Ara-C toxicity in seven patients. The Ara-C concentrations resulting in 50% inhibition of clonogenicity (ID50) were 0.48–123 x 10−8M Ara-C in the absence of stimulatory growth factors, versus only 0.12–0.40 × 10−8M Ara-C in the presence of these factors. In two patients, addition of one or more factors neither increased the number of CFU-L in liquid nor enhanced the Ara-C toxicity. Even in the absence of growth factors the ID50 values in these cases were as low as 0.20 and 0.28 × 10−8M Ara-C and in the same range as the ID50 values observed with maximum growth factor stimulation in the other seven patients. These results indicate that Ara-C cytotoxicity can be enhanced by individually selected, clonogenic cell growth-promoting hematopoietic factors.

Non-selective lymphocyte isolation from human blood by nylon wool filtration and density centrifugation

A new method was employed to isolate lymphocytes from human peripheral blood. Continuous flow filtration through a nylon wool filter, at a flow rate of 1.4 ml/min, produced a lymphocyte yield of 90.5% and a purity of 96% without any shift in the B-T cell ratio. Ficoll-Isopaque with a specific gravity of 1.085 g/ml instead of 1.077 g/ml could be used to remove the erythrocytes. An overall recovery, including defibrination, filtration, Ficoll-Isopaque centrifugation and washing step, of 74.5% was achieved.

Lymphocyte density distribution profile and spontaneous transformation related to the stage of Hodgkin's disease.

. Density distribution profiles of peripheral lymphocytes were made in 41 untreated patients with Hodgkins disease and in 14 normal controls. The fraction of lymphocytes with a low specific gravity in patients was significantly increased in comparison to normal controls (P= 0.0007). These low density lymphocytes in Hodgkins disease patients showed an elevated spontaneous 3H‐thymidine incorporation compared to normal controls (P < 0.0001). In 78% of the patients the spontaneous DNA synthesis of the low density lymphocytes was above the normal range. A positive correlation (P= 0.002) was found between the spontaneous 3H‐thymidine uptake and the stage of the disease.

Cytotoxic potential of anti-CD7 immunotoxin (WT1-ricin A) to purge ex vivo malignant T cells in bone marrow

Summary With the perspective of bone marrow purging in autologous transplantation, we investigated the cytotoxicity of the anti‐T cell immunotoxin (IT) WT1‐ricin A (anti‐CD7) to malignant T cells obtained from patients with T cell acute lymphoblastic leukaemia or lymphoma. The cytotoxic efficacy of IT was based on the extent of protein synthesis inhibition. Cytotoxicity of IT to malignant T cells showed a dependency on antigen density comparable to the T cell lines GH1, CEM, Jurkat, HSB‐2 and HPB‐ALL and was enhanced considerably in the presence of 6 mm ammonium chloride. The ultimate proof of cell kill can only be obtained from clonogenic assays: however, culturing of malignant T cells was not feasible. Therefore these assays were performed with the cell line CEM that expresses comparable amounts of CD7 antigen as malignant T cells of most patients. More than 6‐logs of CEM appeared to be eliminated after incubation with 10‐8m WT1‐ricin A.

DNA strand breaks in human leukocytes induced by chemotherapy and total body irradiation

The occurrence of DNA strand breaks and/or DNA alkali-labile sites in peripheral blood leucocytes was demonstrated ex vivo in three patients during and after bone marrow ablative chemotherapy and total body irradiation (TBI) with use of fluorometric analysis of the DNA unwinding rate in alkaline solution (FADU assay). DNA damage was apparent after cyclophosphamide administration and after TBI, related to the amount of the applied dose. In vivo repair occurred within 24 hours, although not to pretreatment values. Demethoxydaunorubicin and busulfan at the dosages used did not induce measurable DNA strand breaks. The experiences described may be developed further to study ex vivo the occurrence of DNA lesions in patients during and after anticancer treatment. Such studies may be of value in comparing the DNA damaging potential of different chemotherapeutic or radiotherapeutic regimens and as a biological assessment of DNA damage after nuclear casualties in cases where the dose is greater than 1-2 Gy and measurement can be made within due time after the ionizing exposure.

Lymphocyte isolation from human spleen by counterflow centrifugation employing two different flow chambers on line

Studies on splenic lymphocytes have hitherto been performed on single cell suspensions depleted of phagocytic cells by adherence to plastic or incubation with carbonyl iron. These techniques have the disadvantages of selective cell loss, suboptimal cell purification and cell activation. This paper describes purification of splenic lymphocytes by the use of counterflow centrifugation (CFC). The method was adapted to overcome pelleting of cells in the separation chamber to form a plug at the inlet and impede adequate flow. By combining 2 different separation chambers on line in 1 rotor this problem was overcome. Of all lymphocytes recovered after CFC 88.8 +/- 1.4% were collected in 2 pooled fractions with a purity of greater than or equal to 98% and a cell viability of 95%. After CFC, 80.8 +/- 12.1% of the viable cells loaded were recovered.

Cell cycle kinetics of hematopoiesis before and after in vivo administration of GM-CSF in refractory anemia: Evidence for a shortening of the granulocyte release time

SummaryGM-CSF administration to patients with refractory anemia (RA) induces an increase in neutrophils and eosinophils. We studied cell kinetic mechanisms underlying this observation using clonogenic assays and in vivo iododeoxyuridine labeling of bone marrow cells. Cell cycle kinetics were studied in three patients before and during GM-CSF administration (two daily subcutaneous injections of 54 or 108 Μg). No consistent effect on the relative number of bone marrow CFU-GM was noticed. The DNA synthesis time and potential doubling time of low-density bone marrow cells remained essentially the same. A slight decrease (1.5–3.7%) in labeling index was found, originating from the myelo(-mono)cytic lineage. In all three patients the release time of labeled granulocytes from the bone marrow into the peripheral blood was shortened (before GM-CSF treatment 5–7 days and during GM-CSF 3–4 days). Cell cycle kinetics of CD34+ cells were studied in order to obtain kinetic information on immature precursor and progenitor cells. The DNA synthesis time of the CD34+ cells was shortened during GM-CSF therapy, resulting in a shorter potential doubling time. GM-CSF administration to patients with RA results in a rise in granulocytes that might be due partly to an accelerated release of granulocytes from the bone marrow compartment into the circulating blood and partly to an increased proliferative activity of the immature precursor and progenitor cells.

Prophylaxis of Graft Versus Host Disease Using Lymphocyte Depleted Marrow Followed by Short Term Immunosuppression

Graft versus Host Disease (GVHD) is a major complication after allogeneic bone marrow transplantation (BMT), despite the prophylactic use of methotrexate (MTX) or cyclosporin (Cy). Several approaches have been investigated to reduce the incidence and severity of GVHD, postulating that acute GVHD arises from immunologically competent mature T-lymphocytes present in the graft. We have developed a method to remove lymphocytes from marrow grafts by combining density flotation centrifugation and counterflow centrifugation (1). A limitation till now was the small size of the chamber in the Beckman elutriator rotor, which made 5 to 6 consecutive runs of one hour necessary.

Rapid Lymphocyte Depletion by a New Elutriator Rotor for the Prevention of Acute Graft versus Host Disease after Allogeneic Bone Marrow Transplantation

Acute Graft versus Host Disease (GVHD) is responsible for a considerable number of fatalities after allogeneic bone marrow transplantation (BMT). It occurs in 50 to 70 per cent of all adult recipients despite in vitro matching by HLA-analysis and mixed lymphocyte cultures (1). Based on the postulate that acute GVHD arises from immunologically competent mature lymphocytes present in the graft (2), several approaches have been investigated to reduce the incidence and severity of GVHD. We described previously a physical method for the separation of human lymphocytes from committed myeloid and erythroid progenitor cells using density gradient centrifugation followed by counterf low centrifugation (CC) (3). CC provides a rapid and reproducable physical separation technique mainly based on differences in cell size (4). In this study lymphocyte depletion by means of the Beckman single chamber rotor is compared with that attained with a recently developed 4-chamber elutriator rotor. Since the capacity of the rotor has increased 4-fold and the lymphocyte depletion takes place in a closed system, the new rotor saves time and minimizes the risk of infections.