Scand. ]. Immunol., Vol. 5, 1976.
Chronic Lymphocytic Leukemia: Studies on the Effect of Drug Treatment on Different Lymphocytic Subpopulations F. SALSANO, S. S. FR0LAND, J. B. NATVIG & F. MANDELLI First institute of Medical Pathology and Department of Hematology, University of Ron:ie, Rome, Italy, and Institute of Immunology and Rheumatology, Rikshospitalet University Hospital, Oslo, Norway
Salsano, F., Froland, S. S., Natvig, J. B. & Mandelli, F. Chronic Lymphocytic Leukemia: Studies on the Effect of Drug Treatment on Different Lymphocytic Subpopulations. Scand. J. Immunol. 5, 1185-1190, 1976. In the present work the effect of drug treatment on different lymphocyte populations in chronic lymphocytic leukemia was studied. During therapy there was evidence of an increased number of cells unidentifiable by conventional surface markers, provisionally termed lymphocyte surface-marker-negative cells. In addition, evidence of increased numbers of T lymphocytes in untreated patients and further evidence of IgG chronic lymphocytic leukemia cells were obtained. / . B. Natvig, Institute of Immunology and Rheumatology, P. Qvamsgt. 1, Oslo 1, Norway
The concept that chronic lymphocytic leukemia (CLL) represents a monoclonal proliferation of B lymphocytes has been supported by several studies (4, 5, 14, 16) and is partly based on the observations that only one Ig light-chain type is present on the surface membrane of CLL cells and that the same pattern appears after removal by trypsinization and resynthesis of membrane-bound Ig. Furthermore, recent studies of some CLL patients have shown that, when present, serum monoclonal Ig shares idiotypic specificity with membrane Ig and that the idiotypic specificity is shared by IgD and IgM present on the same cells (7, 19). Some problems of the restriction of Ig heavy chains on the membrane of CLL cells are more controversial. Whereas the simultaneous occurrence of jj. and S chains is well established (7, 11, 15), the presence of y chains as membrane Ig on CLL cells, even though reported by most authors (5, 21), is not generally agreed on. It has also been suggested that most IgG-
positive cells in normal peripheral blood are cells with Fc receptors but lacking membrane Ig (24). The aims of this work were to analyze the various lymphocyte populations in CLL by means of membrane Ig and other surface markers and, particularly, to evaluate the effect of drug treatment on different cell populations.
MATERIALS AND METHODS Patients. Fifty-three adult patients with CLL were studied, 37 of them immediately after the diagnosis had been made. Twenty of the untreated patients were also studied 1 month after the start of treatment with either a combination of chlorambucil and prednisone or, alternatively, cyclophosphamide and prednisone. The dosage and schedule of induction and maintenance of this therapy have been reported previously (12). Cells from 16 other CLL patients who
1186 P. Salsano, S. S. Froland, J. B. Natvig & F. Mandelli
had received the same treatment for at least 6 months were also tested. Separation of lymphocytes. Lymphocytes from peripheral venous blood (10 IU/ml of heparin) were obtained by the Isopaque-Ficoll gradient centrifugation method (1) and were subsequently washed three times in Hanks's balanced salt solution (Hanks's BSS). Fluorescein-conjugated antisera. Antisera against human IgG, IgA, IgM, IgD, and the F(ab')2 fragment of human IgG were prepared in rabbits and conjugated with fluorescein isothiocyanate (FITC) as described previously (3, 13). Demonstration of membrane-bound Ig on B lymphocytes. Staining of B lymphocytes (IgC) was performed by incubating 5 X 10^ cells with 0.05-0.01 ml of the FITC-conjugated IgG fraction of the appropriate antiserum with a molar fluorescein to protein (F/P) ratio of 2-3, using a final protein concentration of approximately 1.0 mg/ml, for 30 min at 4°C. Cells were then washed three times in medium 199 (Grand Island Biological Co., New York, N.Y.) containing 1.0 mg/ml of sodium azide. Examination of cells was performed with a LeitE Ortoplan Microscope equipped with a xenoa lamp of 75 W and a vertical illuminator, with a filter combination described elsewhere (3). In the cases where membrane-bound IgG was found on CLL lymphocytes, cells were cultured overnight at 37 °C in serum-free medium. If the IgG disappeared after such a procedure, it was regarded as due to passive uptake from serum. Such cases were not included in the further analysis. Determination of T lymphocytes. T lymphocytes were identified as the percentage of cells forming spontaneous rosettes with sheep erythroq-tes (SRBC). Briefly, 1 X 10" lymphocytes in 0.25 rnl of Hanks's BSS were incubated at 37°C for 5 min with an equal volume of a 1% suspension of washed SRBC, collected not more than 5 days earlier. The mixture was then centiifuged at 200 g for 5 min and reincubated at 4°C overnight. After half of the supernatant had been discarded, the pellet was gently resuspended with a Pasteur pipette, and the per-
Table I. Ig class of the CLL cells in the 53 different CLL patients studied Ig class IgD and IgM IgM JgD IgG
Not classified Total
Untreated* 22 6 4 3 2 37
Treated** H 0 0 3 2 16
Total 33 6 4 6 4 53
* 37 previously untreated patients; 20 of these patients were also tested after 1 month of treatment. ** 16 different patients tested after a treatment period of more than 6 months.
centage of rosette-forming lymphocytes (ERFC) (that is, lymphocytes with three or more erythrocytes firmly bound to the surface) was determined. Determination of lymphocytes luith Fc receptors. The percentage of lymphocytes with receptors for IgG (EA-RFC), a population of lymphocytic cells apparently distinct from T and B lymphocytes (4), was determined by the ability to form rosettes with human erythrocytes sensitized with human anti-Rh antiserum (Ri), as previously described (4, 6). RESULTS Ig classes on CLL cells The distribution of Ig classes among membrane-bound immunoglobulins in the CLL cells is shown in Table I. IgM and IgD were the Ig classes usually found on CLL cells. In the untreated group IgM was found on cells from 28 patients, and in 22 of these IgD was found together with IgM. Similarly, IgM was found associated with IgD in 11 treated patients. Six CLL patients had IgG on leukemic cells, which did not disappear during overnight culture at 37°C, The IgG molecules had thus been synthesized by the CLL cells and represented true mtmhtSine. Ig. Three of these patients were untreated and three were in the treated group. In none of these cases was IgM or IgD demonstrated on the cell membrane.
Drugs and Lymphocyte Subpopulations Table II. Different lymphocyte populations in absolute values (abs) and percentage levels before and after 1 month of treatment* Lymphocyte populations Leukocytes Lymphocytes Ig-C abs. % E-RFC abs. % EA-RFC abs. % Marker-neg. cells abs. %
Before treatment
After treatment
32,829 27,012
10,336 7235
21,771 80.6
4718 65.2
3803 14.1
1308 18.1
232
260
3.6
0.9
1152 15.9
1J77 5.1
* The results are given as mean values in the patients studied. Ig-C = B lymphocytes; E-RFC = lymphocytes forming rosettes with sheep erythrocytes; EARFC ^ lymphocytes forming rosettes with human erythrocytes sensitized with human IgG; marker-neg. cells := lymphocyte surface-marker negative cells.
In two patients from each group, CLL cells gave satisfactory staining with anti-F(ab')2 antiserum, but staining for Ig classes was not adequate because too few cells were available and the test could not be repeated. In Table
1187
I these cases are presented as the "not classified' group. Effect of therapy on three populations of lymphoid cells B lymphocytes (Ig-C). In all 37 untreated patients studied, the percentage of lymphocytes with membrane-bound Ig (Ig-C) detectable by immunofluorescence staining with FITC-labeled anti-F(ab')2 was increased considerably compared with normal adult values ( 5 % 26%) (4, 7); in most patients the percentage was 70%-90% (Table II). A high percentage of Ig-C was also found in the 20 patients when studied after 1 month of therapy, but in these patients the percentage of Ig-C tended to be lower than before treatment (Fig. 1). Comparison of these patients' absolute B lymphocyte counts showed considerably lowec numbers in the group treated for 1 month than in the untreated patients. The differences were statistically significant (P < 0.05). Percentages and absolute numbers of Ig-C in patients with long-term treatment (more than 6 months) were similar to those observed after I month (Fig- 1). r lymphocytes (E-RFC). In the untreated patient group a small percentage of T lymphocytes (E-RFC) was found in all cases (mean, 14.1%; range, l % - 2 8 % ) . However, despite this depression on a percentage basis, the ab-
Fig. 1. Percentages (means) of various lymphocyte populations in untreated (U) and treated (T 1 month and T > 6 months) chronic lymphocytic leukemia (CLL) patients.
Oil — E-RFC; • T
li month)
= EA-RFC;
LJ = marker-neg. cells.
l i e s F. Salsano, S. S. Froland, J. B. Natvig & F. Mandelli
solute numbers of E-RFC were increased in blood from most of these patients (mean, 3803/ mm^) as compared with the values in normal subjects (4). After 1 month of therapy the absolute T-cell values were significantly lower, and the percentages were only slightly increased as compared with values before treatment. In the CLL patients who had received longterm treatment a further absolute decrease of E-RFC was observed. Tije mean of absolute Tcell values (722/mm3) was even lower than in normal individuals, and the percentages were also lower than in the patients with short-term treatment (Fig. 1). In some instances blood cells were first stained for membrane-bound Ig and subsequently allowed to form rosettes with sheep erythrocytes in accordance with standard procedures, with the exception that preincubation at 37°C was omitted. In the patients investigated, very few or no cells ( < 0.05%) were found to have both membrane-bound Ig and the rosette-forming capacity. Fc-receptor-bearing lymphoid cells (EA-RFC). The percentage of lymphoid cells forming rosettes with IgG-sensitized indicator erythrocytes (EA-RFC) was very small in most of the untreated patients (mean, 0.9%). In the two groups of treated patients they were 3.6% and 1.9%, respectively.
negative cells represented up to 75% of the total lymphocytes present. DISCUSSION
The present paper provides evidence that, in CY-h patients during therapy, there is an increased number of cells unidentifiable by conventional surface markers, provisionally termed lymphocyte surface-marker-negative cells. In addition, evidence oi increased numbers of T lymphocytes in untreated patients and further information about IgG leukemias were obtained. Whereas the percentage of T lymphocytes is rnarkedly depressed in most patients with CLL, including those presented in this study, calculations of absolute T-lymphocyte numbers in peripheral blood has shown that these levels are increased or at least normal, even in the presence of large numbers of malignant B lymphocytes. This was clearly demonstrable in our untreated patients, only two of whom had a reduced absolute number of T lymphocytes in blood. The reason for the increase in T lymphocytes in CLL is unknown, but several explanations must be considered. The phenomenon may reflect a specific cell-mediated immune response directed against antigens on leukemic cells. We and others have recently repotted that the membrane-bound immunoglobulins on CLL cells have idiotypic membrane antigens (8, 19). Furthermore, in the mouse, idiotypic antigens on lymphoma cells can funcStudies on lymphocyte surface-marker-negative tion as transplantation antigens in vivo (22). cells Idiotypic antigens on human CLL cells may, When proportions of lymphocytes unac- therefore, stimulate a T-cell response. The incounted for by the three different marker crease in absolute T-cell numbers in blood techniques used (Ig-C, E-RFC, and EA-RFC) from CCL patients might also be due to an were compared in the three patient groups, immunologically unspecific T-cell proliferation the percentages of such lymphocyte surface- induced in some unknown manner by the mamarker-negative cells were found to have in- lignant process, and even a shift of T cells creased during the treatment (Fig. 1.) The from lymphoid tissues to the circulation is differences in the percentage of such cells in possible. the untreated (mean, 5.1%), in the 1-monthControl experiments permitted us to exclude treated (mean, 15.9%), and in long-term- the possibility that the rosette-forming T lymtreated (22.4%) patients were statistically sig- phocytes (E-RFC) were found among the Ignificant (P < 0.05). In some treated patients positive cells. Comparison of T cells in unthe population of lymphocyte surface-marker- treated and in the two groups of treated pa-
Drugs and Lymphocyte Suhpopulations 1189
tients gave the somewhat surprising results that there was no increase in the percentage of T lymphocytes. Absolute T-lymphocyte numbers were either depressed compared with levels in normals or, often, in the lower normal range but were not increased, as might perhaps have been expected as a reflection of the disappearance of Ig-positive cells—that is, CLL cells. The effect of treatment on lymphocyte populations in blood cannot be explained simply by a selective removal of malignant Ig-positive CLL cells, in which case a corresponding increase in proportions of E-RFC would have been the consequence instead of the actual decrease in absolute numbers observed. Therefore, the drug treatment appears to have affected the T-lymphocyte system, either by interference with T-lymphocyte production, by direct destruction of T lymphocytes, or by a shift of circulating T lymphocytes towards the lymphoid tissue compartments. One might also speculate that any specific T-cell stimulation by CLL cell antigens, which may be responsible for increased T-cell levels in untreated patients, might be inhibited during treatment with cytostatic drugs, thus leading to a reduced T-cell proliferation. The origin and identity of the lymphocyte surface-marker-negative cells are not known. They may represent immature cells belonging to lymphocyte populations normally present in blood. Alternatively, they may be malignant cells lacking membrane-bound Ig, possibly because of a greater degree of immaturity in malignant B cells after treatment with cytostatic drugs. If the latter explanation is correct, their elimination by treatment of CLL cells in blood is not at all as impressive as had been thought previously. Comparison of absolute B-lymphocyte counts in the untreated and treated patients showed considerably lower numbers in the treated group. The percentages of B lymphocytes were also lower in the treated than in the untreated group. These findings together with the wellknown reduction of total lymphocyte count induced by treatment in CLL probably reflects the disappearance of many malignant CLL cells from the circulation. Since, however, even
many treated patients still had increased percentages of Ig-C compared with normal values, with a restriction of Ig classes similar to that found on CLL cells from untreated patients, a considerable number of CLL cells probably persist in the circulation even after treatment. The finding of very small numbers of EARFC—lymphocytes with Fc receptors as detected with a rosette technique (4)—confirms Our previous report that CLL cells appear to lack Fc receptors detectable with this test (6). EA-RFC represents the only population with membrane markers which tends to increase in percentage after treatment. The present results extend previous studies on the role of membrane-bound immunoglobulins in B lymphocytes (5, 9, 16, 23) and, in particular, in the malignant B lymphocytes in CLL patients (2, 17). A series of articles has recently presented new information about IgD (10, 18) and IgM (7, 11). These findings have basically been confirmed in the present study. Our study has, however, given additional information on IgG leukemias. Six cases of CLL with IgG-bearing leukemic cells were found, and in all these six cases IgG was present on the CLL lymphocytes in the absence of both IgM and IgD. Nothing definite is known about possible clinical and hematological differences between patients with IgM/IgD-bearing and IgG-bearing CLL cells. However, preliminary findings in our IgG leukemia patients suggest that lymphocyte functions tested in various in vitro systems are better preserved than IgM/IgD in leukemia (20). Further, both our data and analysis of other CLL cases (16) suggest a higher frequency of serum M components in IgG CLL cells than in other CLL forms. This may reflect the origin of IgG-positive CLL cells from more mature lymphoid cells than the IgM/IgD-positive CLL cells.
ACKNOWLEDGEMENTS We are indebted to Dr. Francesca Villa Santa for performance of many rosette tests. The
1190 F. Salsano, S. S. Froland, J. B. Natvig & F. Mandelli
skillful technical assistance of Mrs. Luigina Mazzocchi is gratefully acknowledged. This study was aided by grants from Anders Jahre's Foundation for the Advancement of Science, and the Norwegian Research Council for Science and the Humanities.
REFERENCES 1. Bayum, A. Isolation of lymphocytes, granulocytes and macrophages. Scand. J. Immunoi. 5, Suppl. 5, 9, 1976. 2. Dickler, H. B. & Kunkel, H. G. Interaction of aggregated y-globulin with B lymphocytes. / . exp. Med. 136, 191, 1972. 3. Froland, S. S. & Natvig, J. B. Surface-bound immunoglobulin on lymphocytes from normal and immunodeficient humans. Scand. J. Immunol. 1, 1, 1972. 4. Freland, S. S. & Natvig, J. B. Identification of three different human lymphocyte populations by surface markers. Transplant. Rev. 16, 114, 19735. Fr0land, S. S., Natvig, J. B. & Stavem, P. Immunological characterization of lymphocytes in lymphoprol'ferative disease. Restriction of classes, subclasses, and Gm allotypes of membrane-bound Ig. Scand. J. Immunol. 1, 351, 1972. 6. Fraland, S. S., Wisleff, F. & Michaelsen, T. E. Human lymphocytes with receptors for IgG. A population of cells distinct from T and B lymphocytes. Int. Arch. Allergy 41, 124, 1974. 7. Fu, S. M., Winchester, R. J. & Kunkel, H. G. Occurrence of surface IgM, IgD and free light chains on human lymphocytes. / . exp. Med. 139, 451, 1974. 8. Fu, S. M., Winchester, R. J., Feizi, T., Weher, P. D. & Kunkel, H. G. Idiotypic specificity of surface immunogiobulin and the maturation of leukemic bone-marrow.derived lymphocytes. Proc. nai. Acad. Sci. (USA) 11, 44«7, 1974. 9. Grey, H. M., Rabellino, E. & Pirofsky, B. Immunoglobulins on the surface of lymphocytes. IV. Distribution in hypogammaglobulinemia, cellular immuno deficiency and chronic lymphatic leukemia. / . clin. Invest. 50, 2368, 1971. lO.Knapp, W., Bolhvis, R. L. H., Radl, J. & Higmans, W. Independent movement of IgD and IgM molecules on the surface of individual lymphocytes. /. ImmunoL 111, 1295, 1973. U.Kubo, R. T., Grey, H. M. & Pirofsky, B. IgD: a major immunoglobulin on the surface of lymphocytes from patients with chronic lymphatic leukemia. /. Immtinol. 112, 1952, 1974. 12. Mandelli, F., Salsano, F., De Rossi, G., Fontana, L., Mannella, E., Monarca, B., Pisarri Salsano, S., Received 22 January 1976 Received in revised form 24 September 1976
Tribalto, M. & Villasanta, F. Chronic lymphocytic leukemia (CLL) cells; influence of the therapy on membrane markers, in vitro DNA synthesis and cell-mediated cytotoxicity. in Proceedings of the International Symposium on Hematology, Milan, 1975. In press. 13. Michaelsen, T. E. & Natvig, J. B. Three new fragments, F(ab)2, F(c)2 and Fab/c, obtained by papain proteolysis of normal human IgG. Scand. J. Immunol. 1, 255, 1972. 14. Pernis, B., Ferrarini, M., Forni, L. & Amante, L. Immunoglobulins on lymphocyte membrane, p. 95 in Amos, B. (ed.) Progress in Immunology. Academic Press, New York, 1971. 15. Preud'Homme, J. L., Brouet, J. C , Clauvel, J. P. & Seligmann, M. Surface IgD in immunoproliferative disorders. Scand. j . Immunol. 3, 853, 1974. 16. Preud'Homme, J., L. & Seligmann, M. Surface bound immunoglobulins as a cell marker in human lymphoprol if erative diseases. Blood 40, 777, 1972. 17. Ross, G. D., Rabellino, E. M., Polley, M. J. & Grey, H. (M. Combined studies ol complement receptor and surface immunoglobulin-bearing cells and sheep erythrocyte rosette-forming cells in normal and leukemic human lymphocytes. / . ctiii. Invest. 52, ill, 1973. 18. Rowe, D. S., Hug, K., Forni, L. & Pernis, B. Immunoglobulin D as a lymphocyte receptor. /. exp. Mod. 138, 965, 197>. 19. Salsano, F., Froland, S. S., Natvig, J. B. & Michaelsen, T. E. Same idiotype of B-lymphocyte membrane IgD and IgM. Formal evidence for monoclonality of chronic lymphocytic leukemia cells. Scand. J. Immunol. 3, 841, 1974. 20. Salsano, F., Mannella, E., De Rossi, G., Fontana, L., Pisarri Salsano, S. & Villa Santa, M. F. Popolazioni linfocitarie e terapia nella leucemia linfoide cronica. Progr. Med. 32, 317, 1976. 21. Seligmann, M., Preud'Homme, J. L. & Brouet, J. C. B and T cell markers in human proliferative blood diseases and primary immunodeficiences with special reference to membrane bound immunoglobulins. Transplant. Rev. 16, 85, 1973. 22.Sugai, S. S., Palmer, D. W., Talai, N. & Witg, I. P. Protective and cellular immune response to idiotypic determinants on cells from a spontaneous lymphoma of N2B/N2W Fi mice. /. exp. Med. 140, 1547, 1974. 23. Wilson, J. D. & Nossal, G. J. V. Identification of human T and B lymphocytes in normal peripheral blood and in chronic lymphocytic leukemia. Lancet 2, 788, 1971. 24. Winchester, R. J., Fu, S. M., Hoffman, T. & Kunkel, H. G. IgG on lymphocyte surfaces; technical problems and the significance of a third population. / . Umntinol. 114, 1210, 1975.
Chronic Lymphocytic Leukemia: Studies on the Effect of Drug Treatment on Different Lymphocytic Subpopulations F. SALSANO, S. S. FR0LAND, J. B. NATVIG & F. MANDELLI First institute of Medical Pathology and Department of Hematology, University of Ron:ie, Rome, Italy, and Institute of Immunology and Rheumatology, Rikshospitalet University Hospital, Oslo, Norway
Salsano, F., Froland, S. S., Natvig, J. B. & Mandelli, F. Chronic Lymphocytic Leukemia: Studies on the Effect of Drug Treatment on Different Lymphocytic Subpopulations. Scand. J. Immunol. 5, 1185-1190, 1976. In the present work the effect of drug treatment on different lymphocyte populations in chronic lymphocytic leukemia was studied. During therapy there was evidence of an increased number of cells unidentifiable by conventional surface markers, provisionally termed lymphocyte surface-marker-negative cells. In addition, evidence of increased numbers of T lymphocytes in untreated patients and further evidence of IgG chronic lymphocytic leukemia cells were obtained. / . B. Natvig, Institute of Immunology and Rheumatology, P. Qvamsgt. 1, Oslo 1, Norway
The concept that chronic lymphocytic leukemia (CLL) represents a monoclonal proliferation of B lymphocytes has been supported by several studies (4, 5, 14, 16) and is partly based on the observations that only one Ig light-chain type is present on the surface membrane of CLL cells and that the same pattern appears after removal by trypsinization and resynthesis of membrane-bound Ig. Furthermore, recent studies of some CLL patients have shown that, when present, serum monoclonal Ig shares idiotypic specificity with membrane Ig and that the idiotypic specificity is shared by IgD and IgM present on the same cells (7, 19). Some problems of the restriction of Ig heavy chains on the membrane of CLL cells are more controversial. Whereas the simultaneous occurrence of jj. and S chains is well established (7, 11, 15), the presence of y chains as membrane Ig on CLL cells, even though reported by most authors (5, 21), is not generally agreed on. It has also been suggested that most IgG-
positive cells in normal peripheral blood are cells with Fc receptors but lacking membrane Ig (24). The aims of this work were to analyze the various lymphocyte populations in CLL by means of membrane Ig and other surface markers and, particularly, to evaluate the effect of drug treatment on different cell populations.
MATERIALS AND METHODS Patients. Fifty-three adult patients with CLL were studied, 37 of them immediately after the diagnosis had been made. Twenty of the untreated patients were also studied 1 month after the start of treatment with either a combination of chlorambucil and prednisone or, alternatively, cyclophosphamide and prednisone. The dosage and schedule of induction and maintenance of this therapy have been reported previously (12). Cells from 16 other CLL patients who
1186 P. Salsano, S. S. Froland, J. B. Natvig & F. Mandelli
had received the same treatment for at least 6 months were also tested. Separation of lymphocytes. Lymphocytes from peripheral venous blood (10 IU/ml of heparin) were obtained by the Isopaque-Ficoll gradient centrifugation method (1) and were subsequently washed three times in Hanks's balanced salt solution (Hanks's BSS). Fluorescein-conjugated antisera. Antisera against human IgG, IgA, IgM, IgD, and the F(ab')2 fragment of human IgG were prepared in rabbits and conjugated with fluorescein isothiocyanate (FITC) as described previously (3, 13). Demonstration of membrane-bound Ig on B lymphocytes. Staining of B lymphocytes (IgC) was performed by incubating 5 X 10^ cells with 0.05-0.01 ml of the FITC-conjugated IgG fraction of the appropriate antiserum with a molar fluorescein to protein (F/P) ratio of 2-3, using a final protein concentration of approximately 1.0 mg/ml, for 30 min at 4°C. Cells were then washed three times in medium 199 (Grand Island Biological Co., New York, N.Y.) containing 1.0 mg/ml of sodium azide. Examination of cells was performed with a LeitE Ortoplan Microscope equipped with a xenoa lamp of 75 W and a vertical illuminator, with a filter combination described elsewhere (3). In the cases where membrane-bound IgG was found on CLL lymphocytes, cells were cultured overnight at 37 °C in serum-free medium. If the IgG disappeared after such a procedure, it was regarded as due to passive uptake from serum. Such cases were not included in the further analysis. Determination of T lymphocytes. T lymphocytes were identified as the percentage of cells forming spontaneous rosettes with sheep erythroq-tes (SRBC). Briefly, 1 X 10" lymphocytes in 0.25 rnl of Hanks's BSS were incubated at 37°C for 5 min with an equal volume of a 1% suspension of washed SRBC, collected not more than 5 days earlier. The mixture was then centiifuged at 200 g for 5 min and reincubated at 4°C overnight. After half of the supernatant had been discarded, the pellet was gently resuspended with a Pasteur pipette, and the per-
Table I. Ig class of the CLL cells in the 53 different CLL patients studied Ig class IgD and IgM IgM JgD IgG
Not classified Total
Untreated* 22 6 4 3 2 37
Treated** H 0 0 3 2 16
Total 33 6 4 6 4 53
* 37 previously untreated patients; 20 of these patients were also tested after 1 month of treatment. ** 16 different patients tested after a treatment period of more than 6 months.
centage of rosette-forming lymphocytes (ERFC) (that is, lymphocytes with three or more erythrocytes firmly bound to the surface) was determined. Determination of lymphocytes luith Fc receptors. The percentage of lymphocytes with receptors for IgG (EA-RFC), a population of lymphocytic cells apparently distinct from T and B lymphocytes (4), was determined by the ability to form rosettes with human erythrocytes sensitized with human anti-Rh antiserum (Ri), as previously described (4, 6). RESULTS Ig classes on CLL cells The distribution of Ig classes among membrane-bound immunoglobulins in the CLL cells is shown in Table I. IgM and IgD were the Ig classes usually found on CLL cells. In the untreated group IgM was found on cells from 28 patients, and in 22 of these IgD was found together with IgM. Similarly, IgM was found associated with IgD in 11 treated patients. Six CLL patients had IgG on leukemic cells, which did not disappear during overnight culture at 37°C, The IgG molecules had thus been synthesized by the CLL cells and represented true mtmhtSine. Ig. Three of these patients were untreated and three were in the treated group. In none of these cases was IgM or IgD demonstrated on the cell membrane.
Drugs and Lymphocyte Subpopulations Table II. Different lymphocyte populations in absolute values (abs) and percentage levels before and after 1 month of treatment* Lymphocyte populations Leukocytes Lymphocytes Ig-C abs. % E-RFC abs. % EA-RFC abs. % Marker-neg. cells abs. %
Before treatment
After treatment
32,829 27,012
10,336 7235
21,771 80.6
4718 65.2
3803 14.1
1308 18.1
232
260
3.6
0.9
1152 15.9
1J77 5.1
* The results are given as mean values in the patients studied. Ig-C = B lymphocytes; E-RFC = lymphocytes forming rosettes with sheep erythrocytes; EARFC ^ lymphocytes forming rosettes with human erythrocytes sensitized with human IgG; marker-neg. cells := lymphocyte surface-marker negative cells.
In two patients from each group, CLL cells gave satisfactory staining with anti-F(ab')2 antiserum, but staining for Ig classes was not adequate because too few cells were available and the test could not be repeated. In Table
1187
I these cases are presented as the "not classified' group. Effect of therapy on three populations of lymphoid cells B lymphocytes (Ig-C). In all 37 untreated patients studied, the percentage of lymphocytes with membrane-bound Ig (Ig-C) detectable by immunofluorescence staining with FITC-labeled anti-F(ab')2 was increased considerably compared with normal adult values ( 5 % 26%) (4, 7); in most patients the percentage was 70%-90% (Table II). A high percentage of Ig-C was also found in the 20 patients when studied after 1 month of therapy, but in these patients the percentage of Ig-C tended to be lower than before treatment (Fig. 1). Comparison of these patients' absolute B lymphocyte counts showed considerably lowec numbers in the group treated for 1 month than in the untreated patients. The differences were statistically significant (P < 0.05). Percentages and absolute numbers of Ig-C in patients with long-term treatment (more than 6 months) were similar to those observed after I month (Fig- 1). r lymphocytes (E-RFC). In the untreated patient group a small percentage of T lymphocytes (E-RFC) was found in all cases (mean, 14.1%; range, l % - 2 8 % ) . However, despite this depression on a percentage basis, the ab-
Fig. 1. Percentages (means) of various lymphocyte populations in untreated (U) and treated (T 1 month and T > 6 months) chronic lymphocytic leukemia (CLL) patients.
Oil — E-RFC; • T
li month)
= EA-RFC;
LJ = marker-neg. cells.
l i e s F. Salsano, S. S. Froland, J. B. Natvig & F. Mandelli
solute numbers of E-RFC were increased in blood from most of these patients (mean, 3803/ mm^) as compared with the values in normal subjects (4). After 1 month of therapy the absolute T-cell values were significantly lower, and the percentages were only slightly increased as compared with values before treatment. In the CLL patients who had received longterm treatment a further absolute decrease of E-RFC was observed. Tije mean of absolute Tcell values (722/mm3) was even lower than in normal individuals, and the percentages were also lower than in the patients with short-term treatment (Fig. 1). In some instances blood cells were first stained for membrane-bound Ig and subsequently allowed to form rosettes with sheep erythrocytes in accordance with standard procedures, with the exception that preincubation at 37°C was omitted. In the patients investigated, very few or no cells ( < 0.05%) were found to have both membrane-bound Ig and the rosette-forming capacity. Fc-receptor-bearing lymphoid cells (EA-RFC). The percentage of lymphoid cells forming rosettes with IgG-sensitized indicator erythrocytes (EA-RFC) was very small in most of the untreated patients (mean, 0.9%). In the two groups of treated patients they were 3.6% and 1.9%, respectively.
negative cells represented up to 75% of the total lymphocytes present. DISCUSSION
The present paper provides evidence that, in CY-h patients during therapy, there is an increased number of cells unidentifiable by conventional surface markers, provisionally termed lymphocyte surface-marker-negative cells. In addition, evidence oi increased numbers of T lymphocytes in untreated patients and further information about IgG leukemias were obtained. Whereas the percentage of T lymphocytes is rnarkedly depressed in most patients with CLL, including those presented in this study, calculations of absolute T-lymphocyte numbers in peripheral blood has shown that these levels are increased or at least normal, even in the presence of large numbers of malignant B lymphocytes. This was clearly demonstrable in our untreated patients, only two of whom had a reduced absolute number of T lymphocytes in blood. The reason for the increase in T lymphocytes in CLL is unknown, but several explanations must be considered. The phenomenon may reflect a specific cell-mediated immune response directed against antigens on leukemic cells. We and others have recently repotted that the membrane-bound immunoglobulins on CLL cells have idiotypic membrane antigens (8, 19). Furthermore, in the mouse, idiotypic antigens on lymphoma cells can funcStudies on lymphocyte surface-marker-negative tion as transplantation antigens in vivo (22). cells Idiotypic antigens on human CLL cells may, When proportions of lymphocytes unac- therefore, stimulate a T-cell response. The incounted for by the three different marker crease in absolute T-cell numbers in blood techniques used (Ig-C, E-RFC, and EA-RFC) from CCL patients might also be due to an were compared in the three patient groups, immunologically unspecific T-cell proliferation the percentages of such lymphocyte surface- induced in some unknown manner by the mamarker-negative cells were found to have in- lignant process, and even a shift of T cells creased during the treatment (Fig. 1.) The from lymphoid tissues to the circulation is differences in the percentage of such cells in possible. the untreated (mean, 5.1%), in the 1-monthControl experiments permitted us to exclude treated (mean, 15.9%), and in long-term- the possibility that the rosette-forming T lymtreated (22.4%) patients were statistically sig- phocytes (E-RFC) were found among the Ignificant (P < 0.05). In some treated patients positive cells. Comparison of T cells in unthe population of lymphocyte surface-marker- treated and in the two groups of treated pa-
Drugs and Lymphocyte Suhpopulations 1189
tients gave the somewhat surprising results that there was no increase in the percentage of T lymphocytes. Absolute T-lymphocyte numbers were either depressed compared with levels in normals or, often, in the lower normal range but were not increased, as might perhaps have been expected as a reflection of the disappearance of Ig-positive cells—that is, CLL cells. The effect of treatment on lymphocyte populations in blood cannot be explained simply by a selective removal of malignant Ig-positive CLL cells, in which case a corresponding increase in proportions of E-RFC would have been the consequence instead of the actual decrease in absolute numbers observed. Therefore, the drug treatment appears to have affected the T-lymphocyte system, either by interference with T-lymphocyte production, by direct destruction of T lymphocytes, or by a shift of circulating T lymphocytes towards the lymphoid tissue compartments. One might also speculate that any specific T-cell stimulation by CLL cell antigens, which may be responsible for increased T-cell levels in untreated patients, might be inhibited during treatment with cytostatic drugs, thus leading to a reduced T-cell proliferation. The origin and identity of the lymphocyte surface-marker-negative cells are not known. They may represent immature cells belonging to lymphocyte populations normally present in blood. Alternatively, they may be malignant cells lacking membrane-bound Ig, possibly because of a greater degree of immaturity in malignant B cells after treatment with cytostatic drugs. If the latter explanation is correct, their elimination by treatment of CLL cells in blood is not at all as impressive as had been thought previously. Comparison of absolute B-lymphocyte counts in the untreated and treated patients showed considerably lower numbers in the treated group. The percentages of B lymphocytes were also lower in the treated than in the untreated group. These findings together with the wellknown reduction of total lymphocyte count induced by treatment in CLL probably reflects the disappearance of many malignant CLL cells from the circulation. Since, however, even
many treated patients still had increased percentages of Ig-C compared with normal values, with a restriction of Ig classes similar to that found on CLL cells from untreated patients, a considerable number of CLL cells probably persist in the circulation even after treatment. The finding of very small numbers of EARFC—lymphocytes with Fc receptors as detected with a rosette technique (4)—confirms Our previous report that CLL cells appear to lack Fc receptors detectable with this test (6). EA-RFC represents the only population with membrane markers which tends to increase in percentage after treatment. The present results extend previous studies on the role of membrane-bound immunoglobulins in B lymphocytes (5, 9, 16, 23) and, in particular, in the malignant B lymphocytes in CLL patients (2, 17). A series of articles has recently presented new information about IgD (10, 18) and IgM (7, 11). These findings have basically been confirmed in the present study. Our study has, however, given additional information on IgG leukemias. Six cases of CLL with IgG-bearing leukemic cells were found, and in all these six cases IgG was present on the CLL lymphocytes in the absence of both IgM and IgD. Nothing definite is known about possible clinical and hematological differences between patients with IgM/IgD-bearing and IgG-bearing CLL cells. However, preliminary findings in our IgG leukemia patients suggest that lymphocyte functions tested in various in vitro systems are better preserved than IgM/IgD in leukemia (20). Further, both our data and analysis of other CLL cases (16) suggest a higher frequency of serum M components in IgG CLL cells than in other CLL forms. This may reflect the origin of IgG-positive CLL cells from more mature lymphoid cells than the IgM/IgD-positive CLL cells.
ACKNOWLEDGEMENTS We are indebted to Dr. Francesca Villa Santa for performance of many rosette tests. The
1190 F. Salsano, S. S. Froland, J. B. Natvig & F. Mandelli
skillful technical assistance of Mrs. Luigina Mazzocchi is gratefully acknowledged. This study was aided by grants from Anders Jahre's Foundation for the Advancement of Science, and the Norwegian Research Council for Science and the Humanities.
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