Membrane Differences in Peripheral Blood Lymphocytes from Patients with Chronic Lymphocytic Leukemia and Hodgkin's Disease

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1 Proc. Nat. Acad. Sci. USA Vol. 72, No. 6, pp , June 1975 Membrane Differences in Peripheral Blood Lymphocytes from Patients with hronic Lymphocytic Leukemia and Hodgkin's Disease (concanavalin A/cell agglutination/cell attaclhment/cap formation) URI MINTZ AND LEO SAHS Department of Genetics, Weizmann Institute of Science, Rehovot, Israel; and Department of Oncology, Beilinson Medical enter, Tel-Aviv University Medical School, Israel ommunicated by George Klein, March 3, 1975 ABSTRAT Lymphocytes were isolated from the peripheral blood of 21 normal persons and 66 patients with patients with chronic lymphocytic leukemia (LL) with or peripheral blood lymphocytes of 21 normal persons, 2 chronic lymphocytic leukemia (LL), LL in remission, Hodgkin's disease, Hodgkin's disease in remission, various without treatment, 15 patients with untreated Hodgkin's other tumors, or cardiovascular diseases. The lymphocytes disease, 12 patients with various other tumors, and 1 patients were studied for cap formation and agglutinability by with nonmalignant diseases (cardiovascular diseases). In order concanavalin A, and for cell attachment to the surface of a to determine to what extent patients in clinical remission may petri dish. The frequency of cap formation was lowest in lymphocytes from patients with untreated Hodgkin's disease (2.1 ±.8%), next lowest in lymphocytes from pa- seen in patients with active disease, we have also included six still show surface membrane changes if lymphocytes like those tients with LL who were or were not under treatment patients with Hodgkin's disease in remission and three patients with LL in remission. ( %), and also low in Hodgkin's disease in remission ( %). The frequencies of cap formation by lymphocytes from patients with various other tumors MATERIALS AND METHODS (19.1 ± 2.5%), with LL in remission (24. ±.9%), and with nonmalignant diseases ( %) were more ells. Peripheral blood samples from healthy adult donors similar to the frequency found in lymphocytes from normal persons ( %). Lymphocytes from all the containing 1 units/ml of heparin. All experiments were car- and the different patients were collected into sterile syringes patients, including those in remission, showed a higher degree of agglutinability by concanavalin A than lymphocytes from normal persons. ell attachment to a petri isolated by Ficoll-Hypaque gradient centrifugation (6) and ried out with freshly isolated cells. The lymphocytes were dish was highest with LL, next highest with LL in remission, and low for normal persons and all the other the interphase that contains the lymphocytes were washed the gradients centrifuged for 3 min at 4 X g. ells from patients. Lymphocytes from normal persons that consisted predominantly of thymus-derived cells gave similar three times with phosphate-buffered saline at ph 7.2 and results to isolated normal bone marrow-derived cells. diluted in the same buffer for the experiments. In some experiments, the cells isolated from the gradient were suspended The results indicate that there were different changes in the surface membrane of lymphocytes from patients with in a solution containing iron filings (Technicon Products, New LL, LL in remission, Hodgkin's disease, and Hodgkin's York), incubated for 3 mill at 24 on a lay-adams electrical disease in remission, and that the patients in clinical remission still showed abnormalities in their lymphocytes. rotor at 6 rpm, and the cells with iron filings removed with a magnet. Experiments with two normal persons, two patients The analysis of differences in the surface membrane of normal with LL, and two patients with Hodgkin's disease showed a and malignant cells can be of value in elucidating the mechanism of carcinogenesis and leukemogenesis. Among the mem- induced cap formation, and a slightly lower degree of on A- similar degree of cell attachment and frequency of on A- brane changes that can be analyzed are the interaction of cells induced agglutination, after removal of the cells with iron with various lectins (1, 2) and the ability of cells to attach filings. Bone marrow-derived cells (B cells) from normal to the surface of a petri dish (3, 4). Studies with the lectin persons were separated by using the capacity of thymusderived cells (T cells) to form rosettes with sheep erythrocytes concanavalin A (on A) have shown differences in on A- induced cap formation and agglutinability between normal (7-1). lymphocytes and lymphocytic leukemic cells from mice (1, 2). Experiments on cap formation, agglutinability, and the Patients and Normal Persons. The 2 patients with LL, degree of cell attachment to the surface of a petri dish have also indicated that these are useful criteria for determining changes who had been diagnosed 2 months to 16 years previously, were nine women and 11 men, years old, with leukocyte counts in the surface membrane of human peripheral blood lymphocytes from patients with lymphoproliferative disease (4, 5). of 18,-14, cells/mm3 of which 6-96% were lymphocytes. Seventeen of these patients had received no The present experiments were undertaken to use these treatment for at least 2 years, and the other three were under criteria to detect differences in the surface membranes of daily treatment for 1-2 months with 2-3 mg of prednisone and 2 mg of chlorambucil. The three patients with LL in remission had been diagnosed 5-12 years previously as Abbreviations: LL, chronic lymphocytic leukemia; concanavalin A on A,; F-on A, fluorescein isothiocyanate conju- LL, had been in remission for 4-6 years, and had not been having gated with concanavalin A; B cells, bone marrow-derived cells; under treatment for at least 2 years. They were two women 7T cells, thymus-derived cells. and one man, 6-66 years old, with leukocyte counts of

2 Proc. Nat. Acad. Sci. USA 72 (1975) 83 cells/mm3 of which 24-45% were lymphocytes. Before these patients were in remission, two had not been treated and the third had been treated daily for 1 and 2 months with 1 mg of prednisone and 2 mg of chlorambucil before remission. The 15 patients with Hodgkin's disease were six women and nine men, years old, in stages IIB, IIIA, IHIB, and IVB. The diagnoses were all made after staging laparotomy and the histological patterns were lymphocyte predominance, nodular sclerosis, mixed cellularity, or lymphocyte depletion. All were newly diagnosed patients who had not been treated and who had leukocyte counts of 5,7-23, cells/mm3 of which 3-29% were lymphocytes. The six patients with Hodgkin's disease in remission had been diagnosed as having Hodgkin's disease 15 months to 1.5 years previously and had been in remission for 7 months to 1 years, during which time they had not been under treatment. They were one woman and five men, years old, with leukocyte counts of 4,8-1,4 cells/mm3 of which 26-32% were lymphocytes. Before these patients were in remission, five had been treated with irradiation and one with combination chemotherapy. Of the 12 patients with various malignant tumors, two had carcinoma of the cervix uteri, two had breast carcinoma, two had carcinoma of the lung, one patient each had carcinoma of the stomach, ovary, or endometrium, and one patient each had malignant lymphoma, malignant melanoma, or multiple myeloma. rhese patients were seven women and five men, years old, with leukocyte counts of 5,1-16,5 cells/ mm3 of which 7-39% were lymphocytes. The patients with carcinoma of the stomach, ovary, or endometrium, and with malignant lymphoma had been newly diagnosed after total gastrectomy or hysterectomy in the patients with carcinomas and had not received any chemotherapy or irradiation. The two patients with carcinoma of the breast had been diagnosed 6 years earlier and had received no chemotherapy for 2 months or 1 year. Of the two patients with lung carcinoma, one had been diagnosed 2 months earlier and was receiving daily local irradiation for 5 days a week and the other, diagnosed 16 months earlier, had not been irradiated for the previous 7 months. The two patients with carcinoma of the cervix uteri had been diagnosed 2-3 months previously and were receiving daily local irradiation. The patients with multiple myeloma and malignant melanoma were diagnosed 2 and 14 months previously and were being treated with alkeran or BG (Bacillus almette Guerin), respectively. The 1 patients with nonmalignant diseases were five women and five men, 5-71 years old, with atherosclerotic heart disease, coronary insufficiency, rheumatic heart disease, hypertension, or cerebrovascular accident. All except one were under treatment for at least 6 months with digoxin, propranolol, or methyl-dopa. The leukocyte counts were 5,-9,4 cells/mm3 of which 12-35% were lymphocytes. The 66 patients in the present study include 15 patients with LL, 1 with Hodgkin's disease, and three with LL in remission, used in a previous study (4). The 21 normal persons were three women and 18 men, years old, with leukocyte counts of 4,7-1,2/mm3 of which 27-35% were lymphocytes. on A-Induced ap Formation and Agglutination. Fluorescein isothiosyanate conjugated with on A (F-on A) was obtained from Miles-Yeda, Rehovot. The cells were tested Leukemia and Hodgkin's Disease 2429 for cap formation by incubating with F-on A (1 iug/ml) for 15 min at 37, washing with phosphate-buffered saline, pipetting to dissociate aggregates, and counting the number of cells with a cap in a Zeiss fluorescent microscope. Four hundred cells were counted for each determination and only single cells were counted for calculating the percentage of cells with caps. Similar results were obtained with 1 jtg of F-on A per ml. ells were also incubated with vinblastine sulfate (Eli Lilly and o.) for 3 min at 37 before incubation with F-on A. To test for agglutination, we mixed.5 ml of on A diluted in phosphate-buffered saline to different concentrations with.5 ml of a cell suspension in a 35 mm petri dish (Falcon Plastics no. 18) at a final concentration of 5 X 16 cells per ml. The density and size of aggregates were scored in a scale from - to after a 3 min incubation and shaking at 24 on a lay-adams electrical rotator at 6 rpm. ell Attachment to the Surface of a Petri Dish. ell suspensions (prepared in the same way as for the agglutination assay) of 5 X 16 cells per ml were incubated in 35 mm petri dishes (Falcon Plastics no. 18) for 3 min at 24 with a slight shaking by hand for the first 5 min and then about every 5 min. The amount of cell attachment to the surface of the petri dish was scored in a scale from - to In this scale, ++++ indicated that more than about 75% of the cells were attached (4). No obvious differences were observed between scoring after a light washing of the attached cells with phosphate-buffered saline and scoring without washing so that the results were generally scored without washing. Two to four petri dishes were used for each determination. RESULTS on A-induced cap formation The isolated peripheral blood lymphocytes from 21 normal and 66 patients were tested for on A-induced cap formation. Among these patients were 2 with LL and 15 with Hodgkin's disease. The percentage of T cells in peripheral blood lymphocytes is about 5-8% for normal persons, generally less than 1% for patients with LL, and about 5-7% for patients with Hodgkin's disease (11, 12). A on A-induced cap was seen in 24-36% of the lymphocytes from normal donors which were predominantly T cells and a similar percentage (about 3%) was seen in isolated normal B cells. However, 5-9% of the lymphocytes from LL patients had a cap and only 1-3% of lymphocytes from patients with Hodgkin's disease had a cap (Fig. 1). The 15 patients with Hodgkin's disease had received no treatment. Seventeen of the 2 patients with LL had received no treatment for at least 2 years and three had been under daily treatment for 1-2 months with prednisone and chlorambucil. A similar percentage (5-9%) of cells with a cap was seen with LL patients who were or were not under treatment. ap formation was also tested in lymphocytes from six patients with Hodgkin's disease in remission, three with LL in remission, 12 with various tumors, and 1 with nonmalignant diseases. The various tumors included carcinomas, a multiple myeloma, a malignant melanoma, and a lymphoma. The nonmalignant diseases were cardiovascular diseases. Although 22-24% of the lymphocytes from the patients with LL in remission had a cap, a frequency similar to that found with some normal persons, only 9-13% of lymphocytes from

3 243 Medical Sciences: Mintz and Sachs Proc. Nat. Acad. Sci. USA 72 (1975) 4F-. x 3k " oo 4 a - *9 % * S*. ~~~~~~~ 4) L-) 2k 9 1o. el *. *. Hodgkin's disease Hodgkin's disease in remission LL LL in Various Non-malignant Normal remission tumors diseases FIG. 1. ap formation with fluorescent on A by lymphocytes from normal persons and different types of patients. ells were incubated with fluorescent on A and the percentage of single cells with a cap is shown for each patient and normal person. patients with Hodgkin's disease in remission had a cap. Of the lymphocytes from patients with various tumors and nonmalignant diseases, 16-24% and 24-32% had a cap, respectively (Fig. 1). Peripheral blood lymphocytes from all the normal persons and patients were also incubated with 1, 1, or 1 Mg/ml of vinblastine before incubation with F-on A. Treatment with vinblastine resulted, as in other experiments (4), in about a 1.5- to 2-fold increase in the percentage of cells with a cap and there was a maximum increase at 1 /Ag of vinblastine per ml. This increase by vinblastine did not eliminate the differences in the percentage of cap formation by lymphocytes from patients with LL, Hodgkin's disease, and Hodgkin's disease in remission (Table 1). on A-induced cell agglutination Agglutination with 5-25 &g of on A per ml shows that lymphocytes from all the patients were more highly agglutinable than the lymphocytes from normal persons (Fig. 2). The highest agglutinability was found for patients with LL, Hodgkin's disease, LL in remission, and Hodgkin's disease in remission. Despite the similarity in cap formation by cells from normal persons and from patients with LL in remission, the lymphocytes from patients with LL in remission showed the same high agglutinability as lymphocytes from patients with LL, Hodgkin's disease, and Hodgkin's disease in remission (Fig. 2). ell attachment to the surface of a petri dish Attachment of cells to a petri dish was measured after a 3 min incubation without or with different concentrations of on A (Fig. 3). The results without on A showed a high cell attachment by lymphocytes from patients with LL, an intermediate degree when LL was in remission and a low degree of attachment with lymphocytes from normal persons and all the other patients. The degree of cell attachment was increased by incubation with on A. However, even in the presence of on A, there was still the highest degree of attach- TABLE 1. on A-induced cap formation and agglutinability as well as cell attachment to the surface of a petri dish by lymphocytes from 21 normal persons and 66 patients No. of % ells with capt Agglutination with normal persons With vinblastine on A ell attachment Lymphocytes from* or patients Without vinblastine (1 jsg/ml) (25,g/ml) without on A Normal persons ± It 3.6 +to Nonmalignant diseases to Various tumors ± ± to LL 2 7. it ± LL in remission ±t ± Hodgkin's disease ± ± Hodgkin's disease in remission ±t * The nonmalignant diseases were cardiovascular diseases; the various tumors were carcinomas, a multiple myeloma, malignant melanoma, and lymphoma; LL = chronic lymphocytic leukemia. t Mean ± standard deviation.

4 Proc. Nat. Acad. Sci. USA 72 (1975) Leukemia and Hodgkin's Disease / E Hodgkin's disease / Hodgkin's in remission Various tumors Non-malignant diseases )/ o oncentration of on A(Qg/mt) FIG. 2. Lymphocyte agglutinability with different concentrations of on A. ment with LL and the next highest with LL in remission (Fig. 3). Isolated B lymphocytes from normal persons showed the same low degree of cell attachment and on A agglutinability, as do normal peripheral blood lymphocytes that consist mainly of T cells. DISUSSION The study of peripheral blood lymphocytes from the 21 normal persons and 66 patients with various diseases has shown that the three criteria used (percentage of cells with a on A- induced cap, agglutinability by on A, and cell attachment to the surface of a petri dish) can distinguish peripheral blood lymphocytes from patients with LL with and without treatment as well as from patients with untreated Hodgkin's disease, from lymphocytes from patients with various other tumors, cardiovascular diseases, and T and B lymphocytes from normal persons. The lymphocytes from patients with LL and Hodgkin's disease in clinical remission could also be distinguished from lymphocytes from normal persons (Table 1). The 12 patients with various other tumors included carcinomas, a multiple myeloma, malignant melanoma, and a lymphoma. The average percentage of cells with a cap (19.1 i 2.5%) was lower than that found with cardiovascular diseases (26. i 2.2%) or normal persons (29.4 i 3.8%). Further studies should determine to what extent this somewhat lower degree of cap formation is characteristic of certain types of tumors and the relationship between the different membrane changes and cellular lipids (1, 2, 13, 14). It will also be of interest to study patients with other types of nonmalignant diseases and to determine the possible modifying role of virus infection and serum factors on the membrane changes observed in peripheral blood lymphocytes. The lowest frequency of cap formation was found in Hodgkin's disease, 2.1 i.8% of lymphocytes from patients with Hodgkin's disease had a cap. This may account for the defect in delayed hypersensitivity that has been reported for patients with this disease (15). The presence of some lympho oncentration of on A(Mg/ml) FIG. 3. Attachment of lymphocytes to the surface of a petri dish without or with on A. The lymphocytes were scored for cell attachment after a 3 min incubation at 24. cytes with a cap may be either because some of the abnormal cells had retained the ability to form caps and/or the peripheral blood was contaminated with normal lymphocytes. Treatment of cells with vinblastine can increase the frequency of cap formation with normal lymphocytes (16). The differences in the percentage of lymphocytes with a cap in Hodgkin's disease, Hodgkin's disease in remission, LL, and normal persons, were not abolished by treating the cells with vinblastine. ap formation and agglutination by on A measure different types of mobility of surface receptors for this lectin. ap formation requires large lateral movements of receptors, whereas agglutination requires short-range lateral movements that allow the alignment of receptors for the formation of multiple bridges between cells by on A molecules (17, 18). Lymphocytes from normal persons and patients with LL in remission showed a similar percentage of cells with a cap but a difference in agglutinability. This indicates that these two criteria can help to distinguish lymphocytes which differ in one but not the other type of mobility. It will be of interest to compare the differences in mobility of on A receptors with differences in the mobility of surface antigens (19). The degree of cell attachment to the surface of a petri dish appears to measure a different change in the cell surface. Lymphocytes from patients with LL and Hodgkin's disease both differed from lymphocytes from normal persons, in their greater agglutinability and low frequency of cap formation. However, more lymphocytes from patients with LL attached to the surface of petri dishes than did those from Hodgkin's disease and all the other patients. Our results indicate that the changes in the surface membrane of peripheral blood lymphocytes from LL patients with or without treatment, untreated Hodgkin's disease, LL in remission, and Hodgkin's disease in remission were different

5 2432 Medical Sciences: Mintz and Sachs and that these changes were not found in lymphocytes from patients with various tumors and nonmalignant cardiovascular diseases. The results also indicate that the patients with LL and Hodgkin's disease in clinical remission still showed some abnormalities in their peripheral blood lymphocytes. Note Added in Proof. We have now found that the peripheral blood lymphocytes from seven patients with acute infectious mononucleosis had 4. i.7% cells with a on-a induced cap, a high degree of on A agglutinability, and an intermediate degree of cell attachment. Unlike the lymphocytes from normal persons or any of the other patients studied, the lymphocytes from infectious mononucleosis also showed a high degree of cell aggregation without on A. The changes in the surface membrane of peripheral blood lymphocytes from infectious mononucleosis were, therefore, different from tihe changes found with the other patients. 1. Sachs, L. (1974) in Harvey Lectures (Academic Press, New York), Vol. 68, pp Sachs, L. (1974) in ell Surface in Development, ed. Moscona, A. A. (Wiley Interscience, New York), pp Proc. Nat. Acad. Sci. USA 72 (1976) 3. Fibach, E. & Sachs, L. (1974) J. ell. Physiol. 83, Mintz, U. & Sachs, L. (1975.) Int. J. ancer 15, Ben-Bassat, H., Goldblum, N., Manny, N. & Sachs, L. (1974) Int. J. ancer 14, Boyum, A. (1968) Scand. J. lin. Lab. Invest. 21, Suppl Lay, W. H., Mendes, N. F., Bianco,. & Nussenzweig, V. (1971) Nature 23, Wybran, J., arr, M.. & Fudenberg, H. H. (1972) J. lin. Invest. 51, Froland, S. S. (1972) Scand. J. Immunol. 1, Polliack, A., Lampen, N., larkson, B. D., De Harven, E., Bentwich, Z., Siegal, F. & Kunkel, H. G. (1973) J. Exp. Med. 138, Seligman, M., Preud'homme, J. L. & Brouet, J.. (1973) Transplant. Rea. 16, ohnen, G., Augener, W., Buka, A. & Brittinger, G. (1974) Acta Haematol. 51, Gottfried, E. L. (1967) J. Lipid Res. 8, Vlodavsky, I. & Sachs, L. (1974) Nature 25, Eltringham, J. R. & Kaplan, H. S. (1973) Nat. ancer Inst. Monogr. 35, Edelman, G. M., Yahara, I. & Wang, J. L. (1973) Proc. Nat. Acad. Sci. USA 7, Rutishauser, U. & Sachs, L. (1974) Proc. Nat. Acad. Sci. USA 71, Rutishauser, U. & Sachs, L. (1975) J. ell Biol., in press. 19. Yefenof, E. & Klein, G. (1974) Exp. ell Res. 88,