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Functional abnormalities associated with T lymphocytes from patients with chronic lymphocytic leukemia
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
17, 451-458 (1980)
Functional Abnormalities Associated with T Lymphocytes from Patients with Chronic Lymphocytic Leukemia’ RICHARD T. CALLERY, ANTHONY J. STRELKAUSKAS,~ SAULYANOVICH,~ STANMARKS, DAVIDROSENTHAL, AND STUART F. SCHLOSSMAN Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina, Charleston, South Carolina 29403; and The Sidney Farber Cancer Institute und Peter Bent Brigham Hospital, Boston, Massachusetts 02115
Received March 3, 1980 Purified populations of T and B cells were obtained from individuals with chronic lymphocytic leukemia (CLL) by the use of anti-F(ab’), aflinity column chromatography techniques in which twice the amount of column material used for normal separations was used for the separation of Ig and Ig+ cells from CLL patients. Furthermore, the flow rate was reduced in order to decrease the possibility that some B cells from CLL patients, containing a low density of surface Ig, would pass through without binding to the coupled Sephadex G-200. Examination of surface Ig, P23,30 antigens, and T-cell antigens in the separated populations indicated that both the Ig- and Ig+ populations were highly enriched. These T cells were also tested for their response to mitogenic stimulation as well as their ability to cooperate with leukemic B cells, normal B cells, or B cells from a transformed B-cell line. T cells from leukemic patients were found to be significantly deficient in both the response to mitogens and ability to cooperate with B cells in the synthesis and secretion of immunoglobulin.
INTRODUCTION
It is now well recognized that the majority of cases of chronic lymphocytic leukemia (CLL) are of monoclonal B-cell origin, as determined by cell surface immunoglobulin (Ig) and the presence of Fc receptors (l-5). The functional capabilities of the residual T-cell populations in these patients are still controversial, although the importance of the T-cell component in the prognosis of the disease has been suggested (6). To date, the assessment of T-cell function in patients with B-cell CLL has been extensively investigated by analysis of the response to mitogens. Some investigators have suggested that the residual T cells respond poorly to mitogens (7-9), whereas others have indicated that T cells isolated from CLL patients can respond in a near normal fashion to mitogenic stimulation (10, 11). In earlier studies, we have shown that it is necessary for T cells to cooperate with B cells for maximal synthesis and secretion of Ig (12). It has also been shown that T cells are required for optimal Ig secretion after stimulation with pokeweed 1 Publication No. 355 from the Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina. Research supported in part by USPHS Grants AI-16651, CA06017, CA-09173, CA-19589. AI-12069, and HD-09938. ’ Address correspondence and reprint requests to Dr. A. .I. Strelkauskas, Department of Basic and Clinical Immunology and Microbiology, 171 Ashley Avenue, Charleston, S.C. 29403. J Current address: Medical College of Virginia, Richmond, Va. 23298. 451 0090-1229/80/l 10451-08$01.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved
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mitogen (PWM) (13, 14). We have used a reverse hemolytic plaque assay (15, 16) to study regulation of Ig secretion, in order to define more precisely the functional capabilities of T cells from patients with B-cell CLL. In addition to their own B cells, purified T cells from these patients were also mixed with B cells from normal individuals and from a transformed B-cell line which normally develops increased numbers of Ig-secreting cells when mixed with normal T cells. In all instances, T cells from patients with B-cell CLL were deficient in their capacity to cooperate with CLL B cells, normal B cells, or B cells from an established Ig+ line (SL-T). This lack of cooperation may represent (a) an intrinsic deficiency of T-cell function, or (b) a depletion of one or more functionally distinct T-cell subsets responsible for the regulation of Ig synthesis and secretion. MATERIALS
AND METHODS
Patients. Peripheral blood was obtained prior to any form of therapy from six patients with hematologically diagnosed B-cell chronic lymphocytic leukemia (CLL) (sIg+, Ia+, E-) seen at the Peter Bent Brigham Hospital in Boston. The six cases were staged according to the clinical scheme described by Rai et al. (17) and included four patients with stage 0 (bone marrow and blood lymphocytosis only), one with stage I (lymphocytosis with enlarged nodes), and one with stage III (lymphocytosis with anemia). Isolation of purified T and B lymphocytes from peripheral blood. Peripheral blood was obtained from normal donors and patients with CLL. Lymphocytes were isolated from both groups using Ficoll-Hypaque density gradient centrifirgation. The separated lymphocytes were washed three times in RPM1 1640 medium (Grand Island Biologicals, Grand Island, N.Y.). These unfractionated mononuclear cells were separated into surface Ig+ and Ig- populations as described previously (18). The lymphocytes were adjusted to a concentration of 20 X lo6 cells per ml in RPM1 1640 supplemented with 3.7% EDTA and passed through a Sephadex G-200 anti-F(ab’), column. Cells which were positive for surface Ig adhered. The adherent cells were eluted with a 1% solution of gammaglobulin. This procedure was modified to reduce the possibility that CLL cells with lowdensity surface Ig were not retained on the column, by using twice the amount of column material (20 x lo6 cells per 2 ml) for leukemic cells as was used for normal peripheral blood lymphocytes (20 x lo6 cells per 1 ml) and reducing the flow rate from 1 ml/min to 0.5 ml/min. Characterization of isolated lymphocyte subpopulations. Subpopulations of cells were analyzed for surface Ig with rabbit anti-human Ig (19), T-cell antigens with a rabbit anti-human T-cell reagent which is T-cell specific (20), and for Ia-like antigens (P23,30) (21) known to be present on human B cells. In each case, the antiserum was incubated with cells at dilutions of 1:20 for 1 hr at 4°C. The cells were then washed three times with RPM1 1640 and reacted with human erythrocytes coated with purified goat anti-rabbit Ig. Cell suspensions were pelleted, and the percentage of rosetted cells was determined by counting 200-300 lymphocytes (22). Proliferation of CLL T cells in response to mitogens. The mitogenic response of purified T cells from CLL patients or normal individuals was tested in microcul-
T-
AND
B-CELL
COOPERATION
IN
453
CLL
ture as described previously (18), each using phytohemagglutinin (PHA) (Grand Island Biologicals) or concanavalin A (ConA) (Calbiochem, La Jolla, Calif.) at optimal concentrations of 0.025 pg/ml for PHA and 125 pg/ml for ConA. Mitogen-stimulated cultures were incubated in a 37”C, 5% CO, humid atmosphere for 5 days and pulsed with 0.2 $.Zi C3H]thymidine (1.9 Ci/mmol, Schwartz-Mann, Orangeburg, N.J.) 4 hr prior to harvesting. Cells were washed on a MASH II apparatus (Microbiological Associates, Bethesda, Md.), and the incorporation of [3H]thymidine was measured in a Packard scintillation counter (Packard Instrument Co., Downers Grove, Ill.). Reverse hemolytic plaque assay. Ig-secreting cells were detected as hemolytic plaque-forming cells by a reverse hemolytic plaque assay described previously (12, 15, 16). Briefly, 50 ~1 of a 10% suspension of sheep erythrocytes which had been coated with rabbit anti-human Ig and 50 ~1 of an appropriate dilution of lymphocytes were pipetted into 10 x 75mm glass test tubes containing 0.9 ml of a 0.8% solution of Sea-Plaque agarose (Marine Colloids, Lockland, Maine) and Hanks’ balanced salt solution (HBSS) (Grand Island Biologic&), mixed, and poured into a 60 mm petri dish into which 5 ml of 1% Sea-Kern agarose (Marine Colloids) in HBSS had previously been poured and allowed to solidify. When the top layer had solidified, the petri dishes were placed in an incubator for 1 hr at 37”C, and 1 ml of guinea pig complement (Grand Island Biologicals) diluted at 1: 10 was added. Incubation was continued for an additional hour and the plaques were counted. If necessary, plates were stored at 4°C until counted. RESULTS Characterization Patients with
of Ig- and Ig+ Cells Isolated from Chronic Lymphocytic Leukemia
the Peripheral
Blood of
Ig- cells from six individuals with CLL were isolated by passage over F(ab’)z columns and analyzed for surface Ig and reactivity with anti-T-cell antiserum or antiserum to the P23,30 antigen. Surface Ig+ cells contaminating the Ig- fractions varied from 8 to 17%, with a mean contamination of 10.7% for all six experiments (Table 1). The anti-T-cell antiserum identified a mean of 84.8% of the Ig- fraction. The percentage of Ig- cells identified by anti-P23,30 antiserum varied from 6 to 18% with a mean of 12.8%. This reactivity could be due to null cells present in the Ig- fraction, as well as to sIg- monocytes. Examination of smears of these isolated cells ruled out the possibility that this was the result of granulocyte contamination. Ig+ cells eluted from the F(ab’), columns were characterized using the same parameters as for the Ig- fractions. The percentage of cells in this fraction containing surface Ig varied from 81 to 97%, with a mean of 90% (Table 1). The specific anti-T-cell antiserum identified from 2 to 9% of the cells with a mean of 5%. The anti-P23,30 antiserum identified from 90 to 99% of the Ig+ cell fractions, with a mean of 95.6% of all Ig+ cells tested in the six experiments (Table 1). Mitogenic Response of Purified Lymphocytic Leukemia
T Cells from
Patients
with Chronic
The responses of T cells isolated from three normal donors and three CLL patients to PHA and ConA are shown in Table 2. Values obtained with normal T
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TABLE CHARACTERIZATION
OF Ig-
BLOOD
OF PATIENTS
AND
1
Ig+ CELL
WITH
POPULATIONS
CHRONIC
FROM
LYMPHOCYTIC
THE PERIPHERAL
LEUKEMIA
Ig- population CLL patient 1 2 3 4
5 6
WBC/mm3
Percentage lymphocytes
10,000 38,000 120,000 12,000 118,000 18,000
76 91 90 70 91 51
%Ig+
%T+
10 8 10 17 10 9
83 88 81 82 89 86
Ig+ population
%P23,30+
%Ig+
%T+
90 97 95 81 86 91
4 7
16 14 18 14 6 9
5 2 9 3
%P23,30+ 95 99 99 95 90 96
cells in response to PHA and ConA averaged 49,157, and 26,061 counts per minute, respectively, whereas T cells isolated from CLL patients showed significantly less responsiveness than normal T cells, averaging 19,474 and 6,025 counts per minute with PHA and ConA, respectively. Spontaneous Immunoglobulin Lymphocytes
Synthesis by Normal
and CLL Peripheral
Blood
To determine the number of cells spontaneously secreting Ig, assays were performed on Ficoll-purified lymphocytes from leukemic patients and normal donors. The number of plaque-forming cells per million unfractionated cells from normal donors ranged from 7486 to 9760, averaging 8519 & 947, whereas the number of plaque-forming cells per million cells in the unfractionated leukemic cell populations ranged from 60 to 1230, averaging 464 ? 399 (Table 3). It has been shown that affinity column-purified T cells have virtually no plaque-forming ability, and examination of purified B cells eluted from these columns also show a strikingly reduced ability to hemolize erythrocytes in the reverse hemolytic plaque assay (12). In addition, it has been shown that T cells are
TABLE MITOGENIC
RESPONSES
2
OF PURIFIED
[3H]Thymidine
uptake
T CELLS
(mean
FROM
cpm
CLL
2 SD) in response
Expt
Normal
T cells
CLL
1” 2b 3’
65,626 49,821 32,024
+ 3766 + 5227 f 5413
18,848 25,308 14,266
2, Table 3, Table 5, Table
1. 1. 1.
to
ConA
PHA
a Patient b Patient r Patient
PATIENTS
T cells 2 2113 k 4795 ‘- 924
Normal
T cells
CLL
41,660 14,714 21,809
2 2482 2 1771 + 2296
10,731 4,135 3,211
T cells 2 3105 2 377 k 147
T- AND B-CELL
COOPERATION
455
IN CLL
TABLE 3 PLAQUE-F• RMINGCAPACITYOFNORMALAND CLL PERIPHERALBLOODLYMPHOCYTES Plaque-forming
cells per lo6 cells
CLL (patient)”
Normal 9760 7486 8600 Not done 8233 Not done
1230 (1)
20(2) 300 330 400 466
(3) (4) (5) (6)
n As in Table 1.
required for maximal plaque formation by B cells (12). To assess the effect of normal T cells on leukemic B cells and vice versa, highly purified T- and B-cell populations from leukemic and normal donors were mixed together at ratios of 70% T cells to 30% B cells and assayed for plaque formation. Cells from a transformed Ig+ line (SL-T) were also used to examine T-cell cooperation in Ig secretion (Table 4). In all cases examined, the leukemic T cells were unable to reconstitute the plaque-forming capacity of normal B cells, leukemic B cells, or B cells from the SL-T line. In contrast, normal T cells were able to cooperate with both normal B cells and SL-T B cells, but could not reconstitute Ig secretion in leukemic B-cell populations (Table 4). TABLE4 PLAQUE-F• RMINGCAPACITYOFRECONSTITUTEDPOPULATIONS OFNORMALAND CLL LYMPHOCYTES" Source of T cells CLL patient” 1 2 3 4 5 6 Normal donors 1 2 3 Noneb Noneb Noneb a Patients as in Table 1 b No T cells added.
Plaque-forming cells per lo6 cells in cultures containing B cells from CLL (patient)” 130 (1) 166 (2) 1133 (3) 2203 (4) 900 (5) 2466 (6)
100(2)
313 (3) 1266 (5) 349 (3)
Normals (donor) 1,630 (1) 660(1) 3,600 (2) 1,960 (2) 1,500 (3) 1,366 (3) 11,300 9,910 9,250 3,100
(1) (2) (3) (3)
SL-T B-cell line 1,260 4,093 3,353 330 1,100 300 10,200 8,666 8,750 1,687 -
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DISCUSSION
The data reported here indicate that (a) by modifying the existing methodology we can obtain highly purified populations of T and leukemic B cells from CLL samples, (b) the mitogenic response of these T cells is markedly lower than that observed with normal control T cells, and (c) leukemic T cells exhibit a profound deficiency in their ability to cooperate with leukemic B cells, normal B cells, or Ig+ B cells from a transformed cell line in the synthesis and secretion of Ig. Through the modification of affinity column chromatography for the isolation of purified Ig- T-cell and Ig+ B-cell populations, we were able to isolate highly purified T cells from the peripheral blood of patients with CLL. In order to assess the purity of these isolated populations we used three criteria: (a) reactivity with anti-T-cell antiserum, (b) the percentage of cells bearing surface Ig, and (c) the percentage of cells positive for the P23,30 antigen, which has been shown to be present on the surface of B lymphocytes (21, 23). By doubling the amount of Sephadex used for the purification of normal lymphocytes and by slowing the passage of cells through the Sephadex, we were able to separate a highly purified population of T lymphocytes from those lymphocytes which were shown to be Ig+ and P23,30+. Ig+ B cells were then eluted from the Sephadex using a 1% solution of gammaglobulin. With respect to responsiveness of T cells to mitogens, our data vary from those reported previously (7,8). However, these response values are consistent with the observations of Utsinger (24), who found diminished responsiveness with purified T cells from five CLL patients. The possibility that plasma factors may play a role in the mitogenic responsiveness of leukemic T cells (24), coupled with the fact that there may be a random distribution of unresponsiveness within the population of CLL patients, provided impetus to seek other means to examine the functional capabilities of T cells in the peripheral blood of CLL patients. Since mitogenic responsiveness is, at best, a poorly understood criterion for assessing leukemic cell function, and also in view of the conflicting reports in the literature, we chose to examine a more physiologically relevant function, i.e., the cooperative potential of T cells for the enhanced production of Ig by B cells. We have shown previously that T cells are required for maximum synthesis and secretion of Ig by B cells (12). Consequently, we-used a reverse hemolytic plaque assay to investigate whether or not T cells from CLL patients had the capacity to cooperate with B cells for the production of Ig. In all cases, we found that when these T cells were mixed with B cells from normal or leukemic individuals they did not enhance the synthesis and secretion of Ig. In order to discount the possibility of lack of cooperation due to histoincompatibility between the cell types, we used an Ig+ cell line (SL-T), which we found could cooperate with normal T cells and could thus be used to estimate the cooperative potential of normal T cells from different donors, as judged by increased Ig secretion (unpublished observations). It should be pointed out that this type of cooperation between normal T cells and B-cell lines has been previously documented (25). It should be noted that this enhancement was never equal to that observed with autologous or HLA-identical T and B cell combinations. More importantly, when T cells from CLL patients were mixed with these B cells, Ig secretion was not enhanced, indicating that CLL
T- AND B-CELL
COOPERATION
IN CLL
457
T cells were functionally different from normal T cells with respect to this ‘ ‘helper’ ’ ability. The observed inability of T cells from leukemic individuals to cooperate in the production of Ig could be the result of a number of possibilities, such as a reduction in the numbers of T cells present in the peripheral blood of these patients. There is supportive evidence for this since it has previously been shown that in leukemic individuals who have high levels of E-rosette-forming cells, the Igsecreting capacity is much greater than in leukemic individuals with very low numbers of T cells (26). On the other hand, it is possible that T cells found in leukemic blood may be affected by the “leukemic process” and, like their B-cell counterparts, no longer function normally. A third possibility is that during the disease process, a specific subset of “helper T cells” responsible for the enhancement of Ig production is selectively lost. Interestingly, there is evidence for the existence of such a subset of helper T cells in man (27, 28). Since the experiments described above were designed so that the percentage of T cells in culture would be the same as that found in normal peripheral blood, and yet only minimal cooperation was observed, we feel that the first possible explanation, i.e., a numerical deficiency of T cells, can be ruled out. Consequently, we are left with the possibilities that the lack of cooperation observed is due to either a functional deficiency within the T cells present in these individuals, or the selective loss of a specific subset of helper cells. Experiments to determine whether a subset of helper cells is missing in CLL patients, using subset-specific antisera (28-30), are currently under way in our laboratory. ACKNOWLEDGMENT We thank Charles L. Smith for excellent editorial assistance.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Aisenberg, A. C., and Block, K. L., N. Engl. .I. Med. 287, 272, 1972. Grey, H. M., Rabellino, E., and Pirofsky, B., J. C/in. Invest. 50, 2368, 1971. Preud’homme, J. L., and Seligmann, M., Blood 40, 777, 1972. Shevach, E. M., Heberman, R., Frank, M., and Green, I., J. Clin. Invest. 51, 1933, 1972. Wilson, J. D., and Nossal, G. J. V., Lancet 2, 788, 1971. Catovsky, D., Miliani, E., Okos, A., and Galton, D. A. G., Lance? 2, 751, 1974. Bouroncle, B. A., Blood 34, 166, 1969. Havemann, K., and Rubin, A. D., Proc. Sot. Exp. Bio[. Med. 127, 668, 1968. Smith, J., Cowling, D. C., and Baker, C. R., Lancer 1, 229, 1972. Wybran, J., Chantler, S., and Fudenberg, H. H., Lancet 1, 126, 1973. Fernandez, L. A., MacSween, J. M., and Langley, G. R., Cancer 39, 1168, 1977. Strelkauskas, A. J., Wilson, B. S., Gallery, R. T., Chess, L., and Schlossman, S. F., J. .&p. Med. 146, 1765, 1977. Keightly, R. G., Cooper, M. D., and Lawton, A. R., J. Immunol. 117, 1538, 1976. Fauci, A. S., Pratt, K. R. K., and Whalen, G. J., J. Zmmunol. 117, 2160, 1976. Molinaro, G. A., Maron, E., and Dray, S., Proc. Nat. Acad. Sci. USA 71, 1229, 1974. Eby, W. C., Chong, C. A., Dray, S., and Molinaro, G. A., J. Immunol. 115, 1700, 1975. Rai, K. R., Sawitsky, A., Cronkite, E. P., Chanana, A. D., Levy, R. N., and Paternack, S., Blood 46, 219, 1975. 113, 1113, 1974. Chess, L., MacDermott, R. P., and Schlossman, S. F., J. Immunol. MacDermott, R. P., Chess, L., and Schlossman, S. F., C/in. Immur&. Immunoparhol. 4, 415, 1975.
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20. Yanovich, S., Sallan, S., Harris, K., Lazarus, H., and Schlossman, S. F., Blood 50, (Suppl. l), 233, 1977. 21. Schlossman, S. F., Chess, L., Humphreys, R. E., and Strominger, J. L., Proc. Nat. Acad. Sci. USA 73, 1288, 1976. 22. Strelkauskas, A. J., Teodoreseu, M., and Dray, S., Clin. Exp. Immunol. 22, 62, 1975. 23. Strominger, J. L., Chess, L., Humphreys, R. E., Mann, D., Parham, P., Robb, R., Schlossman, S. F., Springer, T., and Terhorst, C., In “The Role of Products of the Histocompatibility Gene Complex in Immune Responses,” p. 621, Academic Press, New York, 1976. 24. Utsinger, P. D., Blood 46, 883, 1975. 25. Kishimoto, T., Hirano, T., Kuritani, T., Yamamura, Y., Ralph, P., and Good, R., Nature (London)
271,
756, 1978.
26. Strelkauskas, A. J., Eby, W. C., Molinaro, G. A., and Dray, S., Clin. Immunol. Immunopathol. 6, 334, 1976. 27. Strelkauskas, A. J., Schauf, V., Wilson, B. S., Schlossman, S. F., and Chess, L., .I. Immunol. 120, 1278, 1978. 28. Strelkauskas, A. J., Callary, R. T., Bore], Y., and Schlossman, S. F., Clin. Immunol. Zmmunopathol. 14, 47, 1979. 29. Strelkauskas, A. J., and Schlossman, S. F., In “Clinical Immunobiology” (F. Bach and R. A. Good, Eds.), Vol. 4, Academic Press, New York, in press, 1980. 30. Reinherz, E. L., Strelkauskas, A. J., O’Brien, C., and Schlossman, S. F., J. Zmmunol. 123, 83, 1979.
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
17, 451-458 (1980)
Functional Abnormalities Associated with T Lymphocytes from Patients with Chronic Lymphocytic Leukemia’ RICHARD T. CALLERY, ANTHONY J. STRELKAUSKAS,~ SAULYANOVICH,~ STANMARKS, DAVIDROSENTHAL, AND STUART F. SCHLOSSMAN Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina, Charleston, South Carolina 29403; and The Sidney Farber Cancer Institute und Peter Bent Brigham Hospital, Boston, Massachusetts 02115
Received March 3, 1980 Purified populations of T and B cells were obtained from individuals with chronic lymphocytic leukemia (CLL) by the use of anti-F(ab’), aflinity column chromatography techniques in which twice the amount of column material used for normal separations was used for the separation of Ig and Ig+ cells from CLL patients. Furthermore, the flow rate was reduced in order to decrease the possibility that some B cells from CLL patients, containing a low density of surface Ig, would pass through without binding to the coupled Sephadex G-200. Examination of surface Ig, P23,30 antigens, and T-cell antigens in the separated populations indicated that both the Ig- and Ig+ populations were highly enriched. These T cells were also tested for their response to mitogenic stimulation as well as their ability to cooperate with leukemic B cells, normal B cells, or B cells from a transformed B-cell line. T cells from leukemic patients were found to be significantly deficient in both the response to mitogens and ability to cooperate with B cells in the synthesis and secretion of immunoglobulin.
INTRODUCTION
It is now well recognized that the majority of cases of chronic lymphocytic leukemia (CLL) are of monoclonal B-cell origin, as determined by cell surface immunoglobulin (Ig) and the presence of Fc receptors (l-5). The functional capabilities of the residual T-cell populations in these patients are still controversial, although the importance of the T-cell component in the prognosis of the disease has been suggested (6). To date, the assessment of T-cell function in patients with B-cell CLL has been extensively investigated by analysis of the response to mitogens. Some investigators have suggested that the residual T cells respond poorly to mitogens (7-9), whereas others have indicated that T cells isolated from CLL patients can respond in a near normal fashion to mitogenic stimulation (10, 11). In earlier studies, we have shown that it is necessary for T cells to cooperate with B cells for maximal synthesis and secretion of Ig (12). It has also been shown that T cells are required for optimal Ig secretion after stimulation with pokeweed 1 Publication No. 355 from the Department of Basic and Clinical Immunology and Microbiology, Medical University of South Carolina. Research supported in part by USPHS Grants AI-16651, CA06017, CA-09173, CA-19589. AI-12069, and HD-09938. ’ Address correspondence and reprint requests to Dr. A. .I. Strelkauskas, Department of Basic and Clinical Immunology and Microbiology, 171 Ashley Avenue, Charleston, S.C. 29403. J Current address: Medical College of Virginia, Richmond, Va. 23298. 451 0090-1229/80/l 10451-08$01.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved
452
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mitogen (PWM) (13, 14). We have used a reverse hemolytic plaque assay (15, 16) to study regulation of Ig secretion, in order to define more precisely the functional capabilities of T cells from patients with B-cell CLL. In addition to their own B cells, purified T cells from these patients were also mixed with B cells from normal individuals and from a transformed B-cell line which normally develops increased numbers of Ig-secreting cells when mixed with normal T cells. In all instances, T cells from patients with B-cell CLL were deficient in their capacity to cooperate with CLL B cells, normal B cells, or B cells from an established Ig+ line (SL-T). This lack of cooperation may represent (a) an intrinsic deficiency of T-cell function, or (b) a depletion of one or more functionally distinct T-cell subsets responsible for the regulation of Ig synthesis and secretion. MATERIALS
AND METHODS
Patients. Peripheral blood was obtained prior to any form of therapy from six patients with hematologically diagnosed B-cell chronic lymphocytic leukemia (CLL) (sIg+, Ia+, E-) seen at the Peter Bent Brigham Hospital in Boston. The six cases were staged according to the clinical scheme described by Rai et al. (17) and included four patients with stage 0 (bone marrow and blood lymphocytosis only), one with stage I (lymphocytosis with enlarged nodes), and one with stage III (lymphocytosis with anemia). Isolation of purified T and B lymphocytes from peripheral blood. Peripheral blood was obtained from normal donors and patients with CLL. Lymphocytes were isolated from both groups using Ficoll-Hypaque density gradient centrifirgation. The separated lymphocytes were washed three times in RPM1 1640 medium (Grand Island Biologicals, Grand Island, N.Y.). These unfractionated mononuclear cells were separated into surface Ig+ and Ig- populations as described previously (18). The lymphocytes were adjusted to a concentration of 20 X lo6 cells per ml in RPM1 1640 supplemented with 3.7% EDTA and passed through a Sephadex G-200 anti-F(ab’), column. Cells which were positive for surface Ig adhered. The adherent cells were eluted with a 1% solution of gammaglobulin. This procedure was modified to reduce the possibility that CLL cells with lowdensity surface Ig were not retained on the column, by using twice the amount of column material (20 x lo6 cells per 2 ml) for leukemic cells as was used for normal peripheral blood lymphocytes (20 x lo6 cells per 1 ml) and reducing the flow rate from 1 ml/min to 0.5 ml/min. Characterization of isolated lymphocyte subpopulations. Subpopulations of cells were analyzed for surface Ig with rabbit anti-human Ig (19), T-cell antigens with a rabbit anti-human T-cell reagent which is T-cell specific (20), and for Ia-like antigens (P23,30) (21) known to be present on human B cells. In each case, the antiserum was incubated with cells at dilutions of 1:20 for 1 hr at 4°C. The cells were then washed three times with RPM1 1640 and reacted with human erythrocytes coated with purified goat anti-rabbit Ig. Cell suspensions were pelleted, and the percentage of rosetted cells was determined by counting 200-300 lymphocytes (22). Proliferation of CLL T cells in response to mitogens. The mitogenic response of purified T cells from CLL patients or normal individuals was tested in microcul-
T-
AND
B-CELL
COOPERATION
IN
453
CLL
ture as described previously (18), each using phytohemagglutinin (PHA) (Grand Island Biologicals) or concanavalin A (ConA) (Calbiochem, La Jolla, Calif.) at optimal concentrations of 0.025 pg/ml for PHA and 125 pg/ml for ConA. Mitogen-stimulated cultures were incubated in a 37”C, 5% CO, humid atmosphere for 5 days and pulsed with 0.2 $.Zi C3H]thymidine (1.9 Ci/mmol, Schwartz-Mann, Orangeburg, N.J.) 4 hr prior to harvesting. Cells were washed on a MASH II apparatus (Microbiological Associates, Bethesda, Md.), and the incorporation of [3H]thymidine was measured in a Packard scintillation counter (Packard Instrument Co., Downers Grove, Ill.). Reverse hemolytic plaque assay. Ig-secreting cells were detected as hemolytic plaque-forming cells by a reverse hemolytic plaque assay described previously (12, 15, 16). Briefly, 50 ~1 of a 10% suspension of sheep erythrocytes which had been coated with rabbit anti-human Ig and 50 ~1 of an appropriate dilution of lymphocytes were pipetted into 10 x 75mm glass test tubes containing 0.9 ml of a 0.8% solution of Sea-Plaque agarose (Marine Colloids, Lockland, Maine) and Hanks’ balanced salt solution (HBSS) (Grand Island Biologic&), mixed, and poured into a 60 mm petri dish into which 5 ml of 1% Sea-Kern agarose (Marine Colloids) in HBSS had previously been poured and allowed to solidify. When the top layer had solidified, the petri dishes were placed in an incubator for 1 hr at 37”C, and 1 ml of guinea pig complement (Grand Island Biologicals) diluted at 1: 10 was added. Incubation was continued for an additional hour and the plaques were counted. If necessary, plates were stored at 4°C until counted. RESULTS Characterization Patients with
of Ig- and Ig+ Cells Isolated from Chronic Lymphocytic Leukemia
the Peripheral
Blood of
Ig- cells from six individuals with CLL were isolated by passage over F(ab’)z columns and analyzed for surface Ig and reactivity with anti-T-cell antiserum or antiserum to the P23,30 antigen. Surface Ig+ cells contaminating the Ig- fractions varied from 8 to 17%, with a mean contamination of 10.7% for all six experiments (Table 1). The anti-T-cell antiserum identified a mean of 84.8% of the Ig- fraction. The percentage of Ig- cells identified by anti-P23,30 antiserum varied from 6 to 18% with a mean of 12.8%. This reactivity could be due to null cells present in the Ig- fraction, as well as to sIg- monocytes. Examination of smears of these isolated cells ruled out the possibility that this was the result of granulocyte contamination. Ig+ cells eluted from the F(ab’), columns were characterized using the same parameters as for the Ig- fractions. The percentage of cells in this fraction containing surface Ig varied from 81 to 97%, with a mean of 90% (Table 1). The specific anti-T-cell antiserum identified from 2 to 9% of the cells with a mean of 5%. The anti-P23,30 antiserum identified from 90 to 99% of the Ig+ cell fractions, with a mean of 95.6% of all Ig+ cells tested in the six experiments (Table 1). Mitogenic Response of Purified Lymphocytic Leukemia
T Cells from
Patients
with Chronic
The responses of T cells isolated from three normal donors and three CLL patients to PHA and ConA are shown in Table 2. Values obtained with normal T
454
CALLERY
ET AL.
TABLE CHARACTERIZATION
OF Ig-
BLOOD
OF PATIENTS
AND
1
Ig+ CELL
WITH
POPULATIONS
CHRONIC
FROM
LYMPHOCYTIC
THE PERIPHERAL
LEUKEMIA
Ig- population CLL patient 1 2 3 4
5 6
WBC/mm3
Percentage lymphocytes
10,000 38,000 120,000 12,000 118,000 18,000
76 91 90 70 91 51
%Ig+
%T+
10 8 10 17 10 9
83 88 81 82 89 86
Ig+ population
%P23,30+
%Ig+
%T+
90 97 95 81 86 91
4 7
16 14 18 14 6 9
5 2 9 3
%P23,30+ 95 99 99 95 90 96
cells in response to PHA and ConA averaged 49,157, and 26,061 counts per minute, respectively, whereas T cells isolated from CLL patients showed significantly less responsiveness than normal T cells, averaging 19,474 and 6,025 counts per minute with PHA and ConA, respectively. Spontaneous Immunoglobulin Lymphocytes
Synthesis by Normal
and CLL Peripheral
Blood
To determine the number of cells spontaneously secreting Ig, assays were performed on Ficoll-purified lymphocytes from leukemic patients and normal donors. The number of plaque-forming cells per million unfractionated cells from normal donors ranged from 7486 to 9760, averaging 8519 & 947, whereas the number of plaque-forming cells per million cells in the unfractionated leukemic cell populations ranged from 60 to 1230, averaging 464 ? 399 (Table 3). It has been shown that affinity column-purified T cells have virtually no plaque-forming ability, and examination of purified B cells eluted from these columns also show a strikingly reduced ability to hemolize erythrocytes in the reverse hemolytic plaque assay (12). In addition, it has been shown that T cells are
TABLE MITOGENIC
RESPONSES
2
OF PURIFIED
[3H]Thymidine
uptake
T CELLS
(mean
FROM
cpm
CLL
2 SD) in response
Expt
Normal
T cells
CLL
1” 2b 3’
65,626 49,821 32,024
+ 3766 + 5227 f 5413
18,848 25,308 14,266
2, Table 3, Table 5, Table
1. 1. 1.
to
ConA
PHA
a Patient b Patient r Patient
PATIENTS
T cells 2 2113 k 4795 ‘- 924
Normal
T cells
CLL
41,660 14,714 21,809
2 2482 2 1771 + 2296
10,731 4,135 3,211
T cells 2 3105 2 377 k 147
T- AND B-CELL
COOPERATION
455
IN CLL
TABLE 3 PLAQUE-F• RMINGCAPACITYOFNORMALAND CLL PERIPHERALBLOODLYMPHOCYTES Plaque-forming
cells per lo6 cells
CLL (patient)”
Normal 9760 7486 8600 Not done 8233 Not done
1230 (1)
20(2) 300 330 400 466
(3) (4) (5) (6)
n As in Table 1.
required for maximal plaque formation by B cells (12). To assess the effect of normal T cells on leukemic B cells and vice versa, highly purified T- and B-cell populations from leukemic and normal donors were mixed together at ratios of 70% T cells to 30% B cells and assayed for plaque formation. Cells from a transformed Ig+ line (SL-T) were also used to examine T-cell cooperation in Ig secretion (Table 4). In all cases examined, the leukemic T cells were unable to reconstitute the plaque-forming capacity of normal B cells, leukemic B cells, or B cells from the SL-T line. In contrast, normal T cells were able to cooperate with both normal B cells and SL-T B cells, but could not reconstitute Ig secretion in leukemic B-cell populations (Table 4). TABLE4 PLAQUE-F• RMINGCAPACITYOFRECONSTITUTEDPOPULATIONS OFNORMALAND CLL LYMPHOCYTES" Source of T cells CLL patient” 1 2 3 4 5 6 Normal donors 1 2 3 Noneb Noneb Noneb a Patients as in Table 1 b No T cells added.
Plaque-forming cells per lo6 cells in cultures containing B cells from CLL (patient)” 130 (1) 166 (2) 1133 (3) 2203 (4) 900 (5) 2466 (6)
100(2)
313 (3) 1266 (5) 349 (3)
Normals (donor) 1,630 (1) 660(1) 3,600 (2) 1,960 (2) 1,500 (3) 1,366 (3) 11,300 9,910 9,250 3,100
(1) (2) (3) (3)
SL-T B-cell line 1,260 4,093 3,353 330 1,100 300 10,200 8,666 8,750 1,687 -
456
CALLERY
ET
AL.
DISCUSSION
The data reported here indicate that (a) by modifying the existing methodology we can obtain highly purified populations of T and leukemic B cells from CLL samples, (b) the mitogenic response of these T cells is markedly lower than that observed with normal control T cells, and (c) leukemic T cells exhibit a profound deficiency in their ability to cooperate with leukemic B cells, normal B cells, or Ig+ B cells from a transformed cell line in the synthesis and secretion of Ig. Through the modification of affinity column chromatography for the isolation of purified Ig- T-cell and Ig+ B-cell populations, we were able to isolate highly purified T cells from the peripheral blood of patients with CLL. In order to assess the purity of these isolated populations we used three criteria: (a) reactivity with anti-T-cell antiserum, (b) the percentage of cells bearing surface Ig, and (c) the percentage of cells positive for the P23,30 antigen, which has been shown to be present on the surface of B lymphocytes (21, 23). By doubling the amount of Sephadex used for the purification of normal lymphocytes and by slowing the passage of cells through the Sephadex, we were able to separate a highly purified population of T lymphocytes from those lymphocytes which were shown to be Ig+ and P23,30+. Ig+ B cells were then eluted from the Sephadex using a 1% solution of gammaglobulin. With respect to responsiveness of T cells to mitogens, our data vary from those reported previously (7,8). However, these response values are consistent with the observations of Utsinger (24), who found diminished responsiveness with purified T cells from five CLL patients. The possibility that plasma factors may play a role in the mitogenic responsiveness of leukemic T cells (24), coupled with the fact that there may be a random distribution of unresponsiveness within the population of CLL patients, provided impetus to seek other means to examine the functional capabilities of T cells in the peripheral blood of CLL patients. Since mitogenic responsiveness is, at best, a poorly understood criterion for assessing leukemic cell function, and also in view of the conflicting reports in the literature, we chose to examine a more physiologically relevant function, i.e., the cooperative potential of T cells for the enhanced production of Ig by B cells. We have shown previously that T cells are required for maximum synthesis and secretion of Ig by B cells (12). Consequently, we-used a reverse hemolytic plaque assay to investigate whether or not T cells from CLL patients had the capacity to cooperate with B cells for the production of Ig. In all cases, we found that when these T cells were mixed with B cells from normal or leukemic individuals they did not enhance the synthesis and secretion of Ig. In order to discount the possibility of lack of cooperation due to histoincompatibility between the cell types, we used an Ig+ cell line (SL-T), which we found could cooperate with normal T cells and could thus be used to estimate the cooperative potential of normal T cells from different donors, as judged by increased Ig secretion (unpublished observations). It should be pointed out that this type of cooperation between normal T cells and B-cell lines has been previously documented (25). It should be noted that this enhancement was never equal to that observed with autologous or HLA-identical T and B cell combinations. More importantly, when T cells from CLL patients were mixed with these B cells, Ig secretion was not enhanced, indicating that CLL
T- AND B-CELL
COOPERATION
IN CLL
457
T cells were functionally different from normal T cells with respect to this ‘ ‘helper’ ’ ability. The observed inability of T cells from leukemic individuals to cooperate in the production of Ig could be the result of a number of possibilities, such as a reduction in the numbers of T cells present in the peripheral blood of these patients. There is supportive evidence for this since it has previously been shown that in leukemic individuals who have high levels of E-rosette-forming cells, the Igsecreting capacity is much greater than in leukemic individuals with very low numbers of T cells (26). On the other hand, it is possible that T cells found in leukemic blood may be affected by the “leukemic process” and, like their B-cell counterparts, no longer function normally. A third possibility is that during the disease process, a specific subset of “helper T cells” responsible for the enhancement of Ig production is selectively lost. Interestingly, there is evidence for the existence of such a subset of helper T cells in man (27, 28). Since the experiments described above were designed so that the percentage of T cells in culture would be the same as that found in normal peripheral blood, and yet only minimal cooperation was observed, we feel that the first possible explanation, i.e., a numerical deficiency of T cells, can be ruled out. Consequently, we are left with the possibilities that the lack of cooperation observed is due to either a functional deficiency within the T cells present in these individuals, or the selective loss of a specific subset of helper cells. Experiments to determine whether a subset of helper cells is missing in CLL patients, using subset-specific antisera (28-30), are currently under way in our laboratory. ACKNOWLEDGMENT We thank Charles L. Smith for excellent editorial assistance.
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