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Cellular cytotoxic function and potential in acute myelogenous leukaemia
Leukemia Research Vol. 15, No. 7, pp. 641-644, 1991. Printed in Great Britain.
CELLULAR
0145-2126/91 $3.00 + 0.00 Pergamon Press pie
CYTOTOXIC FUNCTION AND POTENTIAL MYELOGENOUS LEUKAEMIA
IN ACUTE
SEAH H. LIM, COLIN P. WORMAN, ANDREW P. JEWELL and ANTHONY H. GOLDSTONE Department of Haematology, University College Hospital, Gower Street, London (Received 8 January 1991. Revision accepted 9 February 1991)
Abstract--Twenty-seven adult AML patients (13 with active disease and 14 in complete remission) were investigated for their cellular cytotoxic potential and function. All AML patients, whether with active disease or in complete remission, showed increased percentage of CD3 + lymphocytes expressing the cytotoxicity-linked cytoplasmic serine esterase, suggesting a higher than normal cytotoxic potential. However, when the cytotoxic function in these patients were analysed in terms of the natural killer and lectin-dependent cellular cytotoxicity, all AML patients, whether with active disease or in complete remission, had impaired target cell lytic activity. This paradox of cytotoxicity is most likely due to the immunosuppressive effect of the serum factor elaborated by leukaemia myeloblasts. Key words: AML, cytotoxic potential, cytotoxic function.
INTRODUCTION
Another group of peptides isolated is the serine esterase enzymes [5, 6] which have been shown to be released during target cell lysis. Although the exact nature by which the serine esterase enzymes exert their cytotoxic activity is yet to be defined, their involvement in cytotoxic effector cell function has been confirmed. Exposure of these cytotoxic cells to specific inhibitors of serine esterase abolishes their cytotoxic capacity [7]. Studies on the cellular cytotoxic function of patients with acute leukaemia have predominantly focused on the NK activity as measured by the lysis of chromium-labelled K562 target cells [8, 9]. These studies have uniformly shown defective functions in these patients at presentation of disease. Very little is however known about the CTL function in these patients. Of more importance is the lack of assessment of the cellular cytotoxic potential in these patients. The feasibility of identification of these cytotoxic peptides by cytochemical staining has provided a means of assessing the cytotoxic potential of these patients. In this paper, we investigated the cellular cytotoxic function of patients with acute myeloid leukaemia in terms of their NK and lectindependent cellular cytotoxic activity, at presentation of disease and when they achieved complete remission following chemotherapy. Used in conjunction with staining for the cytotoxicity-linked cytoplasmic serine esterase enzymes, we also assessed the cellular cytotoxic potential in these patients in order to discover whether the defective cellular cytotoxic function is a result of low cytotoxic potentials in these patients.
CYTOTOXIC T lymphocytes (CTL) and natural killer (NK) cells are potent killers of target cells. Destruction of the target cells by the cellular immune system involves the direct participation of these killer cells. Whilst CTL are restricted in the target they select to kill by recognition of the target antigen in association with the major histocompatibility complex (MHC), NK cells are MHC unrestricted. In vitro assessment of the cellular cytotoxic function has routinely been based on target cell lysis using specific cell lines such as the K562 and Daudi cells. Such an assessment, although useful, only provides information on how cytotoxic these effector cells are under the particular conditions of the experiment. Other indications of the total cytotoxic potential of these cells are unavailable. Recently several cytotoxic peptides have been isolated in the cytoplasmic granules of both CTL and NK cells. The involvement of these granules in lymphocyte-mediated killing was demonstrated primarily by studies which showed that granules isolated from CTL and NK cell clones are cytotoxic [1, 2]. One such peptide is perforin which has been shown to have the capacity to form pores in the surface membrane of target cells [3]. It is immunologically closely related to C9 of the complement system [4]. Correspondence to: Dr Seah H. Lim, Department of Haematology, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, U.K. 641
642
S.H. LIM et al. MATERIALS AND METHODS
Patients and controls
100
80 ¸
_E~ 60-~.
Twenty-seven adult AML patients, 13 with active disease and 14 in complete remission (CR), and 11 normal healthy controls were studied. Peripheral blood was obtained into preservative-free heparinised containers and the mononuclear cells (MNC) were separated by centrifugation of the whole blood on a Ficoll-Hypaque gradient. Low density cells were harvested and washed 3 times in Hanks balanced salt solution (HBSS) containing 0.2% bovine serum albumin before being resuspended in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS). Specimens studied in leukaemic phase were further enriched by rosetting the MNC with AET-treated sheep erythrocytes and further Ficoll-Hypaque centrifugation. The red cell pellet was obtained and the E+ cells isolated by lysis of the sheep erythrocytes with normal human serum. The E+ cells were then washed again with RPMI 1640 supplemented with 10% FCS. Effector cells were only used for cytotoxicity assays if they contained less than 20% myeloblast contamination.
complex and finally with fast red/naphthol ASBI as a chromogenic solution. A 3 min wash in pH 7.2 PBS solution was carried out between each incubation. When the APAAP staining was complete, the slides were counterstained with Harris's hematoxylin.
Cytotoxicity assay
Calculation and statistical methods
51Cr-labelled K562 erythroleukaemia cells were used as targets in a 4-h radio-isotope release assay by the standard method. Briefly, 1 × 104 51Cr-labelled target cells were added in 2 effector:target (E:T) ratios (10:1 and 20:1) with each test in triplicate using U-shaped microtest plate wells. After 4 h of effector/target co-incubation at 37°C, 100 #L of the supernatant was removed and counted for 5~Cr isotope content in a gamma counter. Lectin-dependent cellular cytotoxicity (LDCC) assays were performed under identical conditions with the exception that phytohaemagglutinin (PHA) was added at a concentration of 5 #g/ mL prior to incubation. Spontaneous release was measured by incubating target cells in the absence of effectors and maximum release by exposing target to 0.5% NP40 in distilled water. For all cases, spontaneous release was <10% of maximum release. The percentage of specific lysis was calculated as follows:
The results of the cytotoxicity and serine esterase assays were all expressed as mean-+ S.D. The differences between the various studied groups were calculated using Student's-t-tests.
experimental release - spontaneous release x 100% maximum release spontaneous release -
Serine esterase
This was performed as previously described [10]. Briefly, cytocentrifuged preparations of the MNC were air dried and fixed in 1% paraformaldehyde solution for 30 s. Slides were then transferred into the substrate chromogen solution (2 x 10-4M Na-benzyloxy carbonyl-L-lysine thiobenzyl ester) and 0.16 mg/mL fast blue BB salt (all Sigma reagents) in 0.2 M Tris-HCl, pH 8.1, which was prepared immediately before use. After 15 rain incubation in the substrate chromogen solution at 37°C, slides were washed in PBS pH 7.2 before addition of the immunophenotyping reagents. Immunological markers
The alkaline phosphatase anti-alkaline phosphatase (APAAP) staining technique was used for the consecutive immunophenotyping of the MNC. The mAb used was CD3 (UCHT1, a gift from Professor P.C. Beverley). Briefly, cytocentrifuged slide preparations of the MNC were air dried before incubating for 30 min with primary antibody followed by rabbit anti-mouse Ig, then with the APAAP
• • •
40o~
active AML AMLin CR
Normalcontrol
-
20-
O" 10:1
20:1 E:T ratio
FIG. 1. Natural killer (NK) activity in AML patients.
RESULTS Figure 1 shows the N K cytotoxic activity of the mononuclear cells. Patients studied in leukaemic phase of A M L had severely impaired target lysis when c o m p a r e d to normal controls (p < 0.0005 for E : T ratio of 10 : 1 and p < 0.0005 for E : T ratio of 20:1) (normal controls: E : T 10:1 42.9---24.7%; E : T ratio 20:1 57.1"--21.2%). Although the m e a n % target lysis in these patients were 3.3 - 3.4% for an E : T ratio of 10 : 1 and 7.2 +- 4.7% for an E : T ratio of 20: 1, 10/13 of these patients exhibit a lytic capacity of 3% or less at an E : T ratio of 10: 1 and 9/13 7% or less at an E : T ratio of 20:1. Patients studied in C R of A M L exhibited a higher level of target lysis. The N K activity of these patients were 12.4---8.6% at an E : T ratio of 10:1 and 19.8 - 14.5% at an E : T ratio of 20 : 1. These values are significantly higher than those studied at presentation (p < 0 . 0 0 5 for E : T ratio of 10:1 and p < 0.005 for E : T ratio of 20: 1) but still significantly lower than those obtained in normal healthy controls (p < 0.0005 for E : T ratio of 10: 1 and p < 0.005 for E : T ratio of 20:1). Therefore, although the N K activity of A M L patients improved upon achievement of CR, it was still severely impaired. Figure 2 shows the L D C C activities of A M L patients. The L D C C activities showed similar patterns of impairment to those of the N K activities. At presentation of disease, this lytic capacity was severely impaired when c o m p a r e d to normal controls
AML cytotoxic function and potential
,00t 80 • • •
-->" 6O F~ 40
activeAML AMLin CR Normalcontrol
20
10:1
20:1 E:T ratio
FIG. 2. Lectin-dependent cellular cytotoxicity in AML patients.
100 ¢/)
~ 8o ÷ 0'~ + (0
60
• •
¢'~ 40" ¢D "6
AML patients Normalcontrols
20" 0
FIG. 3. Percentage of CD3+ cells expressing cytotoxicitylinked cytoplasmic serine esterase.
(p < 0.0005 for both E : T ratios of 10:1 and 20:1). However, it improved significantly (p < 0.05 for both E : T ratios of 10:1 and 20: 1) upon achievement of CR but remained lower than normal controls (p < 0.0005 for both E : T ratios of 10:1 and 20: 1). In contrast, measurements of the cytoplasmic cytotoxicity-linked serine esterase in 10 of these patients (3 in leukaemic phase and 7 in CR) showed that there was no difference in the proportions of CD3+ SE+ cells in AML patients, whether studied in CR or in leukaemic phase of the disease. In both cases the numbers of CD3 + SE+ cells were significantly higher than in normal healthy controls (Fig. 3) (p < 0.05), suggesting that there were more CD3+ cells with a cytotoxic potential in AML patients. DISCUSSION In this paper, we have investigated the cellular cytotoxic potential and function of AML patients, both in leukaemic phase and in remission. The cellular cytotoxic potential of these patients was measured in terms of the percentage of mononuclear cells expressing the cytoplasmic cytotoxicity-linked serine esterase while function was measured by the percentage of lysis of erythroleukaemia target cells, both in the presence and absence of phytohaemagglutinin.
643
We have included the lectin-dependent cellular cytotoxicity assay in the assessment of the cellular cytotoxic function because it includes a broader spectrum of lymphocytes with cytotoxic capacity [11, 12] than is demonstrated in the simpler NK assay. In accord with previous studies [8, 9], we have found that AML patients with active disease have severely impaired natural killer activity. In addition, this study also indicates that the lectin-dependent cellular cytotoxicity was also impaired, suggesting that the impairment of the cytotoxic function is a more global one and involved both the natural killer cells and the cytotoxic T lymphocytes. Of more importance are the results of patients studied in remission which showed that upon attainment of remission, the cellular cytotoxic functions improved although they remained significantly lower than in normal healthy individuals. In contrast, results of the serine esterase study indicate that the proportion of lymphocytes expressing this cytotoxic protein was universally higher than normal in AML patients, whether with active disease or in remission. Together with the results of the target lysis, AML patients seem to have a higher than normal cytotoxic potential but disproportionately low cytotoxic function. This paradox of cytotoxicity has never been described before. The reason behind it remains speculative but could have been caused by leukaemia myeloblasts. We have shown in a previous study [13] that adult AML myeloblasts may be immunogenic and capable of inciting a cellular immune response, as evidenced by signs of T-lymphocyte activation such as elevated serum levels of soluble interleukin 2 receptors (slL-2R) amnd CD8. Similar findings together with an elevated serum CD4 levels are a feature of childhood AML [14]. The increased expression of cytoplasmic cytotoxicity-linked serine esterase in our patients is most likely to be the result of T-lymphocyte activation in response to an immunogenic tumour. Such immunosurveillance however may have failed in the presence of tumour-derived suppressor factors which downregulate the cellular cytotoxic function [15]. Hence, although the cytotoxic potential in these patients is upregulated, cytotoxic function remained suppressed, leading to tumour-escape from immunosurveillance. Patients studied in remission exhibited improved, but still lower than normal, cellular cytotoxic function. Whilst we are unable to exclude the possibility that these patients might constitutionally have subnormal cytotoxic potential and thus be predisposed to the development of leukaemia [16], it is more likely that the impaired cytotoxic function was due to the suppressor effector by residual leukaemia
644
S.H. LIMet al.
cells not detected on conventional investigations. Such a suggestion would be further supported by the presence of T-lymphocyte activation in these patients [13]. Our present findings have provided some new insight into the tumour immunology of AML. They indicate that upregulation of the cytotoxic potential may not always be accompanied by a parallel increase in the cytotoxic function. This may explain the relative ineffectiveness of immunotherapy in AML. Whether or not the monitoring of these immunological indices in A M L patients can be of use in predicting prognosis or in the selection of patients for immunotherapy needs further investigation. REFERENCES 1. Henkart P.A. (1985) Mechanism of lymphocytemediated cytotoxicity. Ann. Reo. Immun. 3, 31-58. 2. Podack E.R. (1986) Molecular mechanisms of cytolysis by complement and by cytolytic lymphocytes. J. cell. Biochem. 311, 133-170. 3. Masson D. & Tschopp J. (1985) Isolation of a lytic, pore-forming protein (perforin) from cytolytic T-lymphocytes. J. biol. Chem. 260, 9069-9072. 4. Tschopp J., Masson D. & Stanley K.K. (1986) Structural/functional similarity between proteins involved in complement- and cytotoxic T-lymphocytemediated cytolysis. Nature 322, 831-834. 5. Pasternack M.S., Verret C.R., Liu M.A. & Eisen H.N. (1986) Serine esterase in cytolytic T lymphocytes. Nature 322, 740-743. 6. Masson D. & Tschopp J. (1987) A family of serine esterases in lytic granules of cytolytic T lymphocytes. Cell 49, 679-685. 7. Hudig D., Redelman D. & Minning L.L. (1984) The requirement for proteinase activity for human lymphocyte-mediated natural cytotoxicity (NK): evidence
that the proteinase is serine dependent and has aromatic amino acid specificity of cleavage. J. Immun. 133, 2647-2654. 8. Nasrallah A.G. & Miale T.D. (1983) Decreased natural killer activity in children with untreated acute leukemia. Cancer Res. 43, 5580-5585. 9. Lotzova E., Savary C.A. & Herberman R.B. (1986) Impaired NK cell profile in leukemia patients. In lmmunobiology o f Natural Killer Cells volume II (Lotzova E. & Herberman R.B., Eds), pp. 29-41. Karger, Basel. 10. Wagner L., Goldstone A.H. & Worman C.P. (1989) Demonstration of the increase in serine esterase-positive T cells in Hairy-cell patients undergoing or-interferon therapy. Leukemia 3, 373-378. 11. Phillips J.H. & Lanier L.L. (1986) Lectin-dependent and anti-CD3 induced cytotoxicity are preferentially mediated by peripheral blood cytotoxic T lymphocytes expressing Leu-7 antigen. J. Immun. 136, 1579-1585. 12. Nelson D.L., Bundy B.M., Pitchon H.E., Blaese R.M. & Strober W. (1976) The effector cells in human peripheral blood mediating mitogen-induced cellular cytotoxicity and antibody-dependent cellular cytotoxicity. J. Immun. 117, 1472-1481. 13. Lim S.H., Giles F.J., Worman C.P., Jewell A.P. & Goldstone A.H. (1990) Evidence of T lymphocyte activation in acute myelogenous leukemia (AML). Blood 76 (Suppl. 1), 296a. 14. Piu C.H., Schell M.J., Vodian M.A., Kline S., Mirro J., Crist W.M. & Behm F.G. (1990) Cellular expression and serum levels of CD 4, CD 8, and Interleukin-2 receptor (IL-2R) in childhood acute myeloid leukemia (AML). Blood 76 (Suppl. 1), 310a. 15. Lim S.H., Worman C.P., Jewell A.P. & Goldstone A.H. (1991) Production of tumour-derived suppressor factor in patients with acute myeloid leukaemia. Leukemia Res. 15, 263-268. 16. Lotzova E. (1983) Function of natural killer cells in various biological phenomena. Surv. Synth. Pathol Res. 2, 41-50.
CELLULAR
0145-2126/91 $3.00 + 0.00 Pergamon Press pie
CYTOTOXIC FUNCTION AND POTENTIAL MYELOGENOUS LEUKAEMIA
IN ACUTE
SEAH H. LIM, COLIN P. WORMAN, ANDREW P. JEWELL and ANTHONY H. GOLDSTONE Department of Haematology, University College Hospital, Gower Street, London (Received 8 January 1991. Revision accepted 9 February 1991)
Abstract--Twenty-seven adult AML patients (13 with active disease and 14 in complete remission) were investigated for their cellular cytotoxic potential and function. All AML patients, whether with active disease or in complete remission, showed increased percentage of CD3 + lymphocytes expressing the cytotoxicity-linked cytoplasmic serine esterase, suggesting a higher than normal cytotoxic potential. However, when the cytotoxic function in these patients were analysed in terms of the natural killer and lectin-dependent cellular cytotoxicity, all AML patients, whether with active disease or in complete remission, had impaired target cell lytic activity. This paradox of cytotoxicity is most likely due to the immunosuppressive effect of the serum factor elaborated by leukaemia myeloblasts. Key words: AML, cytotoxic potential, cytotoxic function.
INTRODUCTION
Another group of peptides isolated is the serine esterase enzymes [5, 6] which have been shown to be released during target cell lysis. Although the exact nature by which the serine esterase enzymes exert their cytotoxic activity is yet to be defined, their involvement in cytotoxic effector cell function has been confirmed. Exposure of these cytotoxic cells to specific inhibitors of serine esterase abolishes their cytotoxic capacity [7]. Studies on the cellular cytotoxic function of patients with acute leukaemia have predominantly focused on the NK activity as measured by the lysis of chromium-labelled K562 target cells [8, 9]. These studies have uniformly shown defective functions in these patients at presentation of disease. Very little is however known about the CTL function in these patients. Of more importance is the lack of assessment of the cellular cytotoxic potential in these patients. The feasibility of identification of these cytotoxic peptides by cytochemical staining has provided a means of assessing the cytotoxic potential of these patients. In this paper, we investigated the cellular cytotoxic function of patients with acute myeloid leukaemia in terms of their NK and lectindependent cellular cytotoxic activity, at presentation of disease and when they achieved complete remission following chemotherapy. Used in conjunction with staining for the cytotoxicity-linked cytoplasmic serine esterase enzymes, we also assessed the cellular cytotoxic potential in these patients in order to discover whether the defective cellular cytotoxic function is a result of low cytotoxic potentials in these patients.
CYTOTOXIC T lymphocytes (CTL) and natural killer (NK) cells are potent killers of target cells. Destruction of the target cells by the cellular immune system involves the direct participation of these killer cells. Whilst CTL are restricted in the target they select to kill by recognition of the target antigen in association with the major histocompatibility complex (MHC), NK cells are MHC unrestricted. In vitro assessment of the cellular cytotoxic function has routinely been based on target cell lysis using specific cell lines such as the K562 and Daudi cells. Such an assessment, although useful, only provides information on how cytotoxic these effector cells are under the particular conditions of the experiment. Other indications of the total cytotoxic potential of these cells are unavailable. Recently several cytotoxic peptides have been isolated in the cytoplasmic granules of both CTL and NK cells. The involvement of these granules in lymphocyte-mediated killing was demonstrated primarily by studies which showed that granules isolated from CTL and NK cell clones are cytotoxic [1, 2]. One such peptide is perforin which has been shown to have the capacity to form pores in the surface membrane of target cells [3]. It is immunologically closely related to C9 of the complement system [4]. Correspondence to: Dr Seah H. Lim, Department of Haematology, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, U.K. 641
642
S.H. LIM et al. MATERIALS AND METHODS
Patients and controls
100
80 ¸
_E~ 60-~.
Twenty-seven adult AML patients, 13 with active disease and 14 in complete remission (CR), and 11 normal healthy controls were studied. Peripheral blood was obtained into preservative-free heparinised containers and the mononuclear cells (MNC) were separated by centrifugation of the whole blood on a Ficoll-Hypaque gradient. Low density cells were harvested and washed 3 times in Hanks balanced salt solution (HBSS) containing 0.2% bovine serum albumin before being resuspended in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS). Specimens studied in leukaemic phase were further enriched by rosetting the MNC with AET-treated sheep erythrocytes and further Ficoll-Hypaque centrifugation. The red cell pellet was obtained and the E+ cells isolated by lysis of the sheep erythrocytes with normal human serum. The E+ cells were then washed again with RPMI 1640 supplemented with 10% FCS. Effector cells were only used for cytotoxicity assays if they contained less than 20% myeloblast contamination.
complex and finally with fast red/naphthol ASBI as a chromogenic solution. A 3 min wash in pH 7.2 PBS solution was carried out between each incubation. When the APAAP staining was complete, the slides were counterstained with Harris's hematoxylin.
Cytotoxicity assay
Calculation and statistical methods
51Cr-labelled K562 erythroleukaemia cells were used as targets in a 4-h radio-isotope release assay by the standard method. Briefly, 1 × 104 51Cr-labelled target cells were added in 2 effector:target (E:T) ratios (10:1 and 20:1) with each test in triplicate using U-shaped microtest plate wells. After 4 h of effector/target co-incubation at 37°C, 100 #L of the supernatant was removed and counted for 5~Cr isotope content in a gamma counter. Lectin-dependent cellular cytotoxicity (LDCC) assays were performed under identical conditions with the exception that phytohaemagglutinin (PHA) was added at a concentration of 5 #g/ mL prior to incubation. Spontaneous release was measured by incubating target cells in the absence of effectors and maximum release by exposing target to 0.5% NP40 in distilled water. For all cases, spontaneous release was <10% of maximum release. The percentage of specific lysis was calculated as follows:
The results of the cytotoxicity and serine esterase assays were all expressed as mean-+ S.D. The differences between the various studied groups were calculated using Student's-t-tests.
experimental release - spontaneous release x 100% maximum release spontaneous release -
Serine esterase
This was performed as previously described [10]. Briefly, cytocentrifuged preparations of the MNC were air dried and fixed in 1% paraformaldehyde solution for 30 s. Slides were then transferred into the substrate chromogen solution (2 x 10-4M Na-benzyloxy carbonyl-L-lysine thiobenzyl ester) and 0.16 mg/mL fast blue BB salt (all Sigma reagents) in 0.2 M Tris-HCl, pH 8.1, which was prepared immediately before use. After 15 rain incubation in the substrate chromogen solution at 37°C, slides were washed in PBS pH 7.2 before addition of the immunophenotyping reagents. Immunological markers
The alkaline phosphatase anti-alkaline phosphatase (APAAP) staining technique was used for the consecutive immunophenotyping of the MNC. The mAb used was CD3 (UCHT1, a gift from Professor P.C. Beverley). Briefly, cytocentrifuged slide preparations of the MNC were air dried before incubating for 30 min with primary antibody followed by rabbit anti-mouse Ig, then with the APAAP
• • •
40o~
active AML AMLin CR
Normalcontrol
-
20-
O" 10:1
20:1 E:T ratio
FIG. 1. Natural killer (NK) activity in AML patients.
RESULTS Figure 1 shows the N K cytotoxic activity of the mononuclear cells. Patients studied in leukaemic phase of A M L had severely impaired target lysis when c o m p a r e d to normal controls (p < 0.0005 for E : T ratio of 10 : 1 and p < 0.0005 for E : T ratio of 20:1) (normal controls: E : T 10:1 42.9---24.7%; E : T ratio 20:1 57.1"--21.2%). Although the m e a n % target lysis in these patients were 3.3 - 3.4% for an E : T ratio of 10 : 1 and 7.2 +- 4.7% for an E : T ratio of 20: 1, 10/13 of these patients exhibit a lytic capacity of 3% or less at an E : T ratio of 10: 1 and 9/13 7% or less at an E : T ratio of 20:1. Patients studied in C R of A M L exhibited a higher level of target lysis. The N K activity of these patients were 12.4---8.6% at an E : T ratio of 10:1 and 19.8 - 14.5% at an E : T ratio of 20 : 1. These values are significantly higher than those studied at presentation (p < 0 . 0 0 5 for E : T ratio of 10:1 and p < 0.005 for E : T ratio of 20: 1) but still significantly lower than those obtained in normal healthy controls (p < 0.0005 for E : T ratio of 10: 1 and p < 0.005 for E : T ratio of 20:1). Therefore, although the N K activity of A M L patients improved upon achievement of CR, it was still severely impaired. Figure 2 shows the L D C C activities of A M L patients. The L D C C activities showed similar patterns of impairment to those of the N K activities. At presentation of disease, this lytic capacity was severely impaired when c o m p a r e d to normal controls
AML cytotoxic function and potential
,00t 80 • • •
-->" 6O F~ 40
activeAML AMLin CR Normalcontrol
20
10:1
20:1 E:T ratio
FIG. 2. Lectin-dependent cellular cytotoxicity in AML patients.
100 ¢/)
~ 8o ÷ 0'~ + (0
60
• •
¢'~ 40" ¢D "6
AML patients Normalcontrols
20" 0
FIG. 3. Percentage of CD3+ cells expressing cytotoxicitylinked cytoplasmic serine esterase.
(p < 0.0005 for both E : T ratios of 10:1 and 20:1). However, it improved significantly (p < 0.05 for both E : T ratios of 10:1 and 20: 1) upon achievement of CR but remained lower than normal controls (p < 0.0005 for both E : T ratios of 10:1 and 20: 1). In contrast, measurements of the cytoplasmic cytotoxicity-linked serine esterase in 10 of these patients (3 in leukaemic phase and 7 in CR) showed that there was no difference in the proportions of CD3+ SE+ cells in AML patients, whether studied in CR or in leukaemic phase of the disease. In both cases the numbers of CD3 + SE+ cells were significantly higher than in normal healthy controls (Fig. 3) (p < 0.05), suggesting that there were more CD3+ cells with a cytotoxic potential in AML patients. DISCUSSION In this paper, we have investigated the cellular cytotoxic potential and function of AML patients, both in leukaemic phase and in remission. The cellular cytotoxic potential of these patients was measured in terms of the percentage of mononuclear cells expressing the cytoplasmic cytotoxicity-linked serine esterase while function was measured by the percentage of lysis of erythroleukaemia target cells, both in the presence and absence of phytohaemagglutinin.
643
We have included the lectin-dependent cellular cytotoxicity assay in the assessment of the cellular cytotoxic function because it includes a broader spectrum of lymphocytes with cytotoxic capacity [11, 12] than is demonstrated in the simpler NK assay. In accord with previous studies [8, 9], we have found that AML patients with active disease have severely impaired natural killer activity. In addition, this study also indicates that the lectin-dependent cellular cytotoxicity was also impaired, suggesting that the impairment of the cytotoxic function is a more global one and involved both the natural killer cells and the cytotoxic T lymphocytes. Of more importance are the results of patients studied in remission which showed that upon attainment of remission, the cellular cytotoxic functions improved although they remained significantly lower than in normal healthy individuals. In contrast, results of the serine esterase study indicate that the proportion of lymphocytes expressing this cytotoxic protein was universally higher than normal in AML patients, whether with active disease or in remission. Together with the results of the target lysis, AML patients seem to have a higher than normal cytotoxic potential but disproportionately low cytotoxic function. This paradox of cytotoxicity has never been described before. The reason behind it remains speculative but could have been caused by leukaemia myeloblasts. We have shown in a previous study [13] that adult AML myeloblasts may be immunogenic and capable of inciting a cellular immune response, as evidenced by signs of T-lymphocyte activation such as elevated serum levels of soluble interleukin 2 receptors (slL-2R) amnd CD8. Similar findings together with an elevated serum CD4 levels are a feature of childhood AML [14]. The increased expression of cytoplasmic cytotoxicity-linked serine esterase in our patients is most likely to be the result of T-lymphocyte activation in response to an immunogenic tumour. Such immunosurveillance however may have failed in the presence of tumour-derived suppressor factors which downregulate the cellular cytotoxic function [15]. Hence, although the cytotoxic potential in these patients is upregulated, cytotoxic function remained suppressed, leading to tumour-escape from immunosurveillance. Patients studied in remission exhibited improved, but still lower than normal, cellular cytotoxic function. Whilst we are unable to exclude the possibility that these patients might constitutionally have subnormal cytotoxic potential and thus be predisposed to the development of leukaemia [16], it is more likely that the impaired cytotoxic function was due to the suppressor effector by residual leukaemia
644
S.H. LIMet al.
cells not detected on conventional investigations. Such a suggestion would be further supported by the presence of T-lymphocyte activation in these patients [13]. Our present findings have provided some new insight into the tumour immunology of AML. They indicate that upregulation of the cytotoxic potential may not always be accompanied by a parallel increase in the cytotoxic function. This may explain the relative ineffectiveness of immunotherapy in AML. Whether or not the monitoring of these immunological indices in A M L patients can be of use in predicting prognosis or in the selection of patients for immunotherapy needs further investigation. REFERENCES 1. Henkart P.A. (1985) Mechanism of lymphocytemediated cytotoxicity. Ann. Reo. Immun. 3, 31-58. 2. Podack E.R. (1986) Molecular mechanisms of cytolysis by complement and by cytolytic lymphocytes. J. cell. Biochem. 311, 133-170. 3. Masson D. & Tschopp J. (1985) Isolation of a lytic, pore-forming protein (perforin) from cytolytic T-lymphocytes. J. biol. Chem. 260, 9069-9072. 4. Tschopp J., Masson D. & Stanley K.K. (1986) Structural/functional similarity between proteins involved in complement- and cytotoxic T-lymphocytemediated cytolysis. Nature 322, 831-834. 5. Pasternack M.S., Verret C.R., Liu M.A. & Eisen H.N. (1986) Serine esterase in cytolytic T lymphocytes. Nature 322, 740-743. 6. Masson D. & Tschopp J. (1987) A family of serine esterases in lytic granules of cytolytic T lymphocytes. Cell 49, 679-685. 7. Hudig D., Redelman D. & Minning L.L. (1984) The requirement for proteinase activity for human lymphocyte-mediated natural cytotoxicity (NK): evidence
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