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Lymphokine-activated killer cytotoxicity and lymphocyte subpopulations in patients with acute leukemia
~Pergamon
Leukemia Research Vol. 18, No. 11, pp. 815-822, 1994 Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0145 2126/94 $7.00 + 0.00
0145-2126(94)00093-X LYMPHOKINE-ACTIVATED KILLER CYTOTOXICITY AND LYMPHOCYTE SUBPOPULATIONS IN PATIENTS WITH ACUTE LEUKEMIA Antonio Parrado, Sofia Casares and Juan-Manuel Rodriguez-Fernfindez Servicio de Hematologla y Hemoterapia, Hospital Universitario Virgen del Rocio, Servicio Andaluz de Salud, Sevilla, Spain (Received 15 February 1994. Revision accepted 18 July 1994) Abstract--In this report we investigated lymphokine-activated killer (LAK) cytotoxicity and lymphocyte subpopulations of peripheral blood mononuclear cells (PBMCs) of patients with acute leukemia in complete remission after chemotherapy or autologous bone marrow transplantation (ABMT) and of normal donors. A positive linear correlation was found between the percentage of spontaneous LAK activity and that of lymphocyte subpopulations with phenotypes CD56 +, CD3-CD8 + CD3-CD56 ÷ and CD3 CD57 ÷ in both groups of patients. A 3day culture with IL-2 produced an up-regulation in the expression of the CD25, CD69, and HLADR markers proportional to the LAK cytotoxicity levels generated in the culture. Determination of percentages of spontaneous and in vitro generated LAK activity as well as of the abovementioned phenotypic markers contribute to the analysis of the process of LAK activation in patients with acute leukemia and may also be useful in those cases in which immunotherapy with IL-2 and/or LAK cells is anticipated. Key words: LAK cells, acute leukemia, chemotherapy, ABMT, cytotoxicity, flow cytometry.
the NK-resistant targets by MC without incubation with IL-2 is termed spontaneous LAK activity. In vitro generated LAK cells in patients with acute leukemia and in controls are able to lyse to a certain degree the allogeneic and autologous leukemic blasts [8-19], thus supporting the use of IL-2 and/or LAK cells as treatment against residual minimal leukemia. We have previously observed that the percentages of NK activity developed by peripheral blood mononuclear cells (PBMC) over the K562 cell line are related to the percentages of lymphocyte population with CD3-CD16 +, CD3 CD56 + and CD3-CD8 + phenotypes in patients with acute leukemia in complete remission (CR) and in normal individuals [20]. In this study we found correlations between the percentages of some lymphocyte subpopulations defined by monoclonal antibodies (MoAb) and the percentages of spontaneous LAK activity measured over the Raji cell line in the PBMCs of patients with acute leukemia in CR after chemotherapy (CT) and autologous bone marrow transplantation (ABMT). Moreover, we have determined how some phenotypic markers are modulated in the in vitro generation of LAK activity.
Introduction Lymphokine-activated killer (LAK) activity is a form of cytotoxicity generated from non-preimmunized mononuclear cells (MC) when submitted to shortterm culture with IL-2 [1]. Most LAK cells seem to arise from natural killer (NK) cells, although T-cells may also play a role [2-5]. Recognition and lysis mechanisms developed by NK and LAK cells do not imply the involvement of the major histocompatibility complex (MHC). LAK activity appears when using autologous or allogeneic target tumor cells resistant to the NK activity [6, 7]. The cytotoxic activity exerted over Abbreviations: LAK, lymphokine-activated killer; PBMC, peripheral blood mononuclear cells; ABMT, autologous bone marrow transplantation; 1L-2, interleukin 2; MC, mononuclear cells; NK, natural killer; MHC, major histocompatibility complex; MoAb, monoclonal antibodies; CR, complete remission; CT, chemotherapy; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; FITC, fluorescein-isothiocyanate; PE, phycoerythrin; E: T, effector to target ratio; CI, confidence interval; PBL, peripheral blood lymphocytes. Correspondence to: Dr Antonio Parrado, Servicio de Hematologia y Hemoterapia, Hospital Universitario Virgen del Rocio, Avda. Manuel Siurot, s/n, 41013 Sevilla, Spain (Fax: (5)4248124).
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Patients
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Materials and Methods
70
We studied 31 patients with acute leukemia in CR postCT, including 14 cases with acute lymphoblastic leukemia (ALL) and 17 with acute myeloid leukemia (AML); 53 patients with acute leukemia post-ABMT, including 29 ALL and 24 AML. Fourteen normal donors served as a control group. PBMCs were obtained by standard Ficoll density gradient centrifugation from normal donors, from post-CT patients 0-27 months (median 8 months) after the end of the induction therapy and from post-ABMT patients 0-43 months (median 9 months) after ABMT.
Generation of LA K cells PBMCs from 29 patients in the post-CT group (13 ALL and 16 AML) and from nine normal donors were cultured for 3 days at a concentration of 10 6 cells/ml in RPMI 1640 medium with 5% human AB serum (complete medium) and 500U/ml natural IL-2 obtained from human lymphocyte cultures (Biotest Pharma GmbH, Dreieich, Germany). PBMCs cultured in parallel for 3 days in complete medium without the addition of IL-2 served as controls.
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Cytotoxicity assay The human lymphoid Raji was used as a target to measure LAK activity as previously described [8]. Briefly, 5 X 10 6 Raji cells were labeled by incubation with 100 ~tCi 51Cr for 90 rain. In triplicate, 5 x 103 target (T) cells and varying numbers of effector (E) cells at E : T ratios of 2.5 : 1, 10:1, 40:1 and 80:1 suspended in RPMI-1640 medium containing 5% heat-inactivated human AB serum were incubated together in 96-well round-bottomed microtiter plates for 4 h. Control wells containing target cells alone in the medium or 2% sodium dodecyl sulphate (SDS) were used to measure spontaneous release (SR) and maximum release (MR) of chromium. The supernatants were collected using the Skatron Supernatant Collection System and measured in a gamma counter. The percentage of experimental lysis was calculated according to the following formula: % lysis = 100 x (ER - SR)/(MR - SR), where ER represents experimental release of SaCr.
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Lymphocyte subpopulations Surface marker analysis was performed in a FACScan which utilized the CONSORT 32 system and the software application LYSYS II (Becton-Dickinson, San Jose, CA) using the following MoAb: anti-CD2 (Leu-5b), CD3 (Leu4), CD4 (Leu-3), CD8 (Leu-2), CD16 (Leu-ll), CD19 (Leu-12), CD20 (Leu-16), CD25, CD56 (Leu-19), CD57 (Leu-7), CD69 (Leu-23), HLA-DR, HLA-DQ, TCRoLfl, TCRy6 (Becton Dickinson, Mountain View, CA) and polyclonal anti-kappa and anti-lambda antibodies (Dakopatts, Glostrup, Denmark). We performed double labeling with fluorescein-isothiocyanate (FITC) and phycoerythrin (PE) conjugated MoAb in the following combinations: CD2/ CD3, CD3/CD4, CD3/CD8, CD4/CD8, TCRo:fl/CD3, TCRafl/CD4, TCRolfl/CD8, TCR76/CD3, TCRy6/CD4,
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Fig. 1. Correlation of spontaneous LAK activity and lymphocyte subpopulation CD56 + for: (A) post-CT; and (B) post-ABMT patients. Spontaneous LAK activity was measured against Raji cell line at E : T ratio of 80:1; r represents Pearson's correlation coefficient; p is the significance of r; a low p-value implies that the data have a significant linear trend.
Statistical analysis" Correlations were calculated with the simple linear regression analysis. Differences between groups were confirmed using Student's t-test. Two-tailed p values of 0.05 or less were considered significant. Results
Relationship between spontaneous L A K activity and lymphocyte subpopulations Significant linear correlations were found between the percentages of cytolytic activity of the PBMCs over the Raji cell line and the percentages of lymphocyte subpopulations with phenotype CD56 + (Fig. 1), C D 3 - C D 8 + (Fig. 2), C D 3 - C D 5 6 + (Fig. 3) and C D 3 - C D 5 7 + (Fig. 4) in both patient groups (postCT and post-ABMT). A m o n g the controls, the correlations between spontaneous L A K activity and subpopulations C D 3 - C D 8 + (r = 0.531, p = 0.05) and CD3 CD56 + (r = 0.469, p = 0.09) were close to the level of significance. Correlations between the spontaneous L A K activity and the subpopulations CD3 C D I 6 ÷ (r = 0.497, p = 0.000), CD16+CD56 + (r = 0.358, p = 0.009), CD16+CD57 + (r = 0.367, p = 0.007) and CD56+CD57 + (r = 0.423, p = 0.002) were only found for the p o s t - A B M T group. Mean percentages of spontaneous L A K activity were 5.6-+ 7.5 and 8.2-+ 9.9 for the post-CT group at
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Fig. 4. Correlation of spontaneous LAK activity and lymphocyte subpopulation CD3-CD57 + for: (A) post-CT; and (B) post-ABMT patients. Spontaneous LAK activity was measured against Raji cell line at E : T ratio of 80 : 1.
Relationship between in vitro generated LAK activity and lymphocyte subpopulations
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the control group, respectively. At E : T of 80 : 1,5 out of 31 (16.1%) post-CT patients overcame confidence limits at 95% for spontaneous L A K activity (Confidence interval (CI), 4.7-11.6%), as well as 13 out of 53 (24.5%) post-ABMT patients (CI, 6.2-11.9%), and 1 out of 14 (7.1%) controls (CI, 0.6-11.7%).
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Fig. 2. Correlation of spontaneous LAK activity and lymphocyte subpopulation CD3-CD8 + for: (A) post-CT; and (B) post-ABMT patients. Spontaneous LAK activity was measured against Raji cell line at E : T ratio of 80 : 1.
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Fig. 3. Correlation of spontaneous LAK activity and lymphocyte subpopulation CD3 CD56 + for: (A) post-CT; and (B) post-ABMT patients. Spontaneous LAK activity was measured against Raji cell line at E : T ratio of 80 : 1.
E : T of 40:1 and 80:1, respectively, 6.4 + 8.4 and 9 . 1 + 10.5 for the p o s t - A B M T at the same E : T ratios, respectively, and 4.6 _+ 6.5 and 6.1 _+ 9.6 for
Mean percentages of lytic activity over the Raji cell line and of the different lymphocyte subpopulations before and after culture with and without IL-2 are shown in Tables 1 and 2. Initial percentages of lysis were not correlated with those obtained after incubation with IL-2. Significant percentage increases were observed in the cultures without IL-2 in relation to the initial values for the subpopulations CD3 + (p = 0.0005), CD4 + (p = 0.0006) and TCRcq3 + (p = 0.02) in the post-CT group and for CD3 + (p = 0.008), CD4 + (p = 0.0003), TCRcrfl + ( p = 0 . 0 1 ) , CD69 + ( p = 0.04), H L A - D R + (p = 0.01) and C D 3 + C D 8 + (p = 0.008) in the control group. Significant percentage decreases were observed in these cultures for the subpopulations CD16 + (p = 0.05), H L A - D R + (p = 0.004), C D 3 - C D 1 6 + (p = 0.003), C D 3 - C D 5 6 + (p = 0.002) and CD16+CD56 + (p = 0.01) in the post-CT group and for CD3-CD8* (p = 0.0009),
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A. Parrado et al. Table 1. P e r c e n t a g e (mean_+ S.D.) of lymphokine-activated killer activity in p o s t - C T p a t i e n t s and n o r m a l d o n o r s b e f o r e (day 0) and after culture (day 3) without or with IL-2 P o s t - C T patients
Normal donors
E:T
- I L - 2 (0)
- I L - 2 (3)
+ I L - 2 (3)
- I L - 2 (0)
- I L - 2 (3)
+ I L - 2 (3)
2.5 : 1 10:1 40:1 80:1
2.5 -+ 3.0 3.2 -+ 4.8 5.9_+7.7 8.5+10.1
0.7 + 1.3 1.5 -+ 2.1 2.6_+3.1 4.1_+4.1
10.4 -+ 14.3 22.0 _+ 20.4 35.7-+25.6 43.5-+25.5
1.4 + 1.7 1.2 + 1.7 3.0-+2.2 3.9-+3.1
0.4 -+ 1.3 0.6 -+ 1.7 1.6_+4.3 2.6-+6.6
8.8 -+ 4.2 24.7 -+ 8.8 47.1_+11.9 54.2-+8.5
Table 2. P e r c e n t a g e ( m e a n -+- S . D . ) of l y m p h o c y t e subpopulations in p o s t - C T patients and normal d o n o r s b e f o r e (day 0) and after culture (day 3) without or with IL-2 Post-CT patients - I L - 2 (0) CD3 + CD4 + CD8 + CD4+CD8 + CD4+/CD8 + TCRa~fl + TCRy6 + CD2 + CD16 + CD56 + CD57 + CD25 + CD69 + HLA-DR + HLA-DQ + CD19 + CD20 + SIgK÷ sIg;. + CD3+CD8 + CD3-CD8 + CD3+CD16 + CD3-CD16 + CD4+CD16 + CD8+CD16 + CD3+CD56 + CD3-CD56 + CD4+CD56 + CD8+CD56 + CD3+CD57 ÷ CD3-CD57 + CD4+CD57 + CD8+CD57 + CD16+CD56 + CD16+CD57 + CD56+CD57 +
74.8 35.1 31.0 2.4 1.44 67.1 8.8 84.5 21.7 31.8 24.3 7.3 17.7 26.1 6.4 6.4 6.6 2.9 1.5 26.0 4.9 11.7 10.1 2.9 7.4 18.9 12.8 5.2 11.5 18.6 5.7 3.4 13.0 14.5 9.1 11.3
-+ 13.9 _+ 12.6 _ 12.5 _+ 2.8 _+ 1.18 + 15.3 + 7.9 -+ 10.3 -+ 11.6 +- 12.2 + 15.3 + 3.2 -+ 10.8 -+ 13.9 -+ 4.6 + 6.0 -+ 5.0 --+ 3.5 --+ 2.2 -+ 12.9 + 4.6 _+ 8.8 -+ 7.8 -+ 2.4 -+ 8.0 -+ 9.0 -+ 9.0 -+ 4.9 -+ 7.9 -+ 14.3 + 5.4 -+ 2.7 +- 9.9 -+ 10.4 -+ 8.8 +- 8.4
C D 3 C D 1 6 + ( p = 0.03) and C D 3 in t h e c o n t r o l g r o u p .
- I L - 2 (3) 87.5 48.3 32.7 2.6 1.94 77.2 10.1 88.7 15.1 24.6 19.8 6.6 16.1 19.7 5.7 3.5 5.0 1.1 0.6 29.5 3.2 10.9 4.6 4.1 5.5 17.8 5.4 6.5 10.8 16.4 3.4 4.0 11.2 8.4 5.6 7.6
-+ 10.5 _+ 15.5 _+ 13.4 _+ 3.0 _+ 1.59 + 13.5 -+ 9.7 -+ 11.1 +- 9.9 -+ 11.1 +- 12.8 -+ 4.2 +- 8.8 +- 10.0 -+ 4.0 -+ 3.9 -+ 3.9 --+ 1.7 --+ 1.1 -+ 13.6 -+ 4.3 -+ 9.1 -+ 3.8 -+ 2.8 -+ 3.9 -+ 8.3 -+ 5.1 -+ 3.2 + 7.3 -+ 12.6 -+ 4.4 -+ 2.8 -+ 8.7 -+ 5.0 +- 7.2 + 5.2
C D 5 6 + ( p = 0.01)
S i g n i f i c a n t p e r c e n t a g e i n c r e a s e s w e r e o b s e r v e d in t h e c u l t u r e s w i t h I L - 2 in r e l a t i o n t o t h e initial v a l u e s
Normal d o n o r s + I L - 2 (3) 87.3 _+ 10.5 45.1 _+ 14.9 41.5 _+ 14.3 5.7 _+ 4.9 1.38 _+ 1.13 78.1 -+ 11.3 12.3 + 6.3 93.2 +- 5.3 17.1 +- 8.8 30.9 -+ 13.3 26.1 -+ 13.3 16.8 -+ 11.8 38.4 -+ 19.2 39.3 -+ 16.5 16.3 -+ 11.5 3.9 + 2.9 4.8 -+ 3.9 1.2--+ 1.5 0.6 --+ 0.9 37.1 -+ 14.9 4.2 +- 4.1 12.0 + 7.2 5.1 -+ 5.4 4.1 -+ 2.9 7.3 + 4.6 22.7 -+ 12.2 8.3 -+ 7.2 8.4 -+ 5.4 15.3 -+ 10.1 21.9 -+ 12.4 4.2 +- 5.1 6.0 -+ 4.6 16.9 -+ 11.9 12.4 -+ 7.5 7.7 -+ 6.0 11.6 -+ 8.4
- I L - 2 (0) 72.6 40.2 29.8 1.1 1.40 66.4 3.4 82.1 19.4 27.1 23.1 7.7 10.6 17.6 6.1 7.2 7.1 3.1 2.1 22.0 7.8 6.4 13.0 1.7 7.3 13.1 14.0 2.9 10.8 15.0 8.1 2.6 15.1 15.2 9.9 11.8
_+ 9.4 _+ 4.1 _+ 7.8 _+ 2.1 _+ 0.25 -+ 9.1 + 2.5 -+ 8.0 + 5.6 -+ 5.4 -+ 9.4 -+ 1.8 -+ 7.6 + 7.8 -+ 3.7 -+ 3.7 -+ 2.2 --+ 2.0 --+ 1.3 -+ 8.5 +- 1.8 _+ 5.2 -+ 5.1 _+ 1.8 _+ 1.5 -+ 7.5 _+ 5.5 -+ 2.3 -+ 3.5 _+ 10.5 -+ 4.8 -+ 1.7 -+ 8.9 -+ 5.5 -+ 4.7 -+ 4.1
- I L - 2 (3) 86.4 49.3 36.1 2.0 1.41 79.7 5.2 88.8 18.3 29.1 21.7 7.4 21.1 28.2 7.7 5.6 6.8 1.7 1.1 32.7 3.4 11.6 6.8 4.1 6.9 22.8 6.3 8.9 12.0 16.1 5.6 3.1 16.2 13.7 8.3 10.1
_+ 10.2 + 4.4 _+ 6.5 _+ 1.5 _+ 0.29 -+ 10.6 -+ 3.1 -+ 7.0 -+ 7.6 -+ 11.6 + 9.3 +- 3.1 -+ 12.2 -+ 7.6 -+ 3.6 -+ 3.1 -+ 2.8 _+ 1.7 + 0.9 -+ 6.2 +- 2.6 -+ 9.2 -+ 5.6 -+ 3.1 -+ 2.5 -+ 14.9 -+ 5.6 -+ 7.4 +- 3.3 -+ 9.1 -+ 4.7 -+ 1.4 + 8.5 +- 5.8 -+ 4.0 +- 3.9
+ I L - 2 (3) 86.1 _+ 5.1 51.6 _+ 7.4 39.8 -+ 8.7 3.4 _+ 2.6 1.37 + 0.41 78.7 -+ 6.0 5.6 -+ 2.5 89.6 -+ 4.8 19.4 -+ 8.2 29.4 + 10.2 22.8 -+ 10.7 13.3 +- 4.6 38.9 +- 13.6 43.7 +- 16.5 13.8 -+ 4.9 6.(1 -+ 2.7 6.3 -+ 2.9 1.9 +-- 1.9 1.8 _+ 1.4 34.9 -+ 7.0 4.9 _+ 2.7 12.6 -+ 7.2 6.9 -+ 2.6 4.6 -+ 3.1 8.2 -+ 4.1 21.8 -+ 10.7 7.7 -+ 3.5 8.1 -+ 5.6 13.8 -+ 4.5 18.2 -+ 11.4 4.6 -+ 1.3 5.0 +- 2.5 18.3 -+ 11.1 13.1 -+ 5.6 7.9 -+ 3.3 11.2 -+ 3.0
b o t h in p o s t - C T a n d c o n t r o l g r o u p s f o r t h e s u b p o p u l a t i o n s C D 3 + ( p = 0.0003 a n d p - - 0.0000, respectively), C D 4 + ( p = 0.007 a n d p = 0.0000), C D 8 + ( p = 0 . 0 0 4 a n d p = 0 . 0 0 0 1 ) , TCRa~fi + ( p =
LAK cytotoxicity and lymphocyte subpopulations
819
Table 3. PBMC and PBL counts (× 10 ~ ml) and percentage of PBL (mean -+ S.D.) in post-CT patients and normal donors before (day 0) and after culture (day 3) without or with IL-2 Normal donors
Post-CT patients
PBMC counts PBL percentages PBL counts
- I L - 2 (0)
- I L - 2 (3)
+IL-2 (3)
1000 -+ 0 70.8 -+ 17.8 708 -+ 178
632 -+ 205 85.9 +- 16.4 535 -+ 194
643 +- 233 92.2 +- 11.0 594 -+ 227
- I k - 2 (0)
- I L - 2 (3)
+IL-2 (3)
1000 -+ 0 81.6 +- 7.5 816 +- 75
848 +- 277 87.8 +- 6.5 756 -+ 262
770 +- 287 96.7 +- 6.4 752 +- 305
Table 4. Absolute counts (x 10 -3 ml, mean +- S.D.) of lymphocyte subpopulations in post-CT patients and normal donors before (day 0) and after culture (day 3) without or with IL-2 Normal donors
Post-CT patients
CD8 + CD4+CD8 + CD25 + CD69 + HLA-DR + HLA-DQ + CD3+CD8 *
- I L - 2 (0)
- I L - 2 (3)
+IL-2 (3)
- I L - 2 (0)
- I L - 2 (3)
+IL-2 (3)
222 -+ 112 17 _+ 24 50 -+ 22 126 + 88 181 -+ 105 43 -+ 30 188 -+ 112
175 + 103 13 -+ 16 33 -+ 21 87 _+ 58 103 + 71 32 -+ 30 159 _+ 103
244 +- 139 32 -+ 30 101 -+ 88 225 + 162 220 -+ 108 89 -+ 58 221 +- 140
242 +_ 69 9 +- 17 63 -+ 18 86 +- 63 145 _+ 68 51 +- 34 179 +- 75
258 13 57 180 211 63 237
287 +- 131 22 _+ 16 101 +_ 58 301 _+ 191 334 _+ 221 109 +- 62 253 -+ 112
0.003 and p = 0.004), C D 2 + (p = 0.0002 and p = 0.03), C D 2 5 + (p = 0.0001 and p = 0.004), C D 6 9 + (p = 0.0000 and p = 0.0001), H L A - D R + (p = 0.003 and p = 0.0006), H L A - D Q + ( p = 0.0000 and p = 0.002), C D 3 + C D 8 + (p = 0.005 and p = 0.003) and C D 4 + C D 8 + ( p = 0.002 and p = 0.05). Significant p e r c e n t a g e decreases were o b s e r v e d in these cultures for the subpopulations CD16 + (p = 0.04), C D 3 - C D 1 6 + (p = 0.009) and C D 1 6 + C D 5 6 + (p = 0.008) in the p o s t - C T g r o u p and for C D 3 - C D 8 + (p = 0.02), C D 3 - C D 1 6 + ( p = 0.005) and C D 3 C D 5 6 + (p = 0.01) in the control group. A joint analysis of these results shows the existence of p e r c e n t a g e increases in the subpopulations C D 8 +, C D 2 5 +, C D 6 9 +, H L A - D R +, H L A - D Q +, C D 3 + C D 8 ÷ and C D 4 + C D 8 + in cultures with IL-2 in relation to the initial values and those o b t a i n e d after culture w i t h o u t IL-2. A f t e r 3 days of culture without and with IL-2, cell counts suffered significant decreases. L y m p h o c y t e s were enriched in culture, and absolute l y m p h o c y t e counts suffered less p r o n o u n c e d decreases (Table 3). A b s o l u t e counts of the relevant l y m p h o c y t e subpopulations are s h o w n in Table 4. Significant increases in culture with IL-2 were observed for C D 2 5 + ( p = 0.005 for p o s t - C T g r o u p ) , C D 6 9 + (p = 0.0000 a n d p = 0.005 for p o s t - C T and control groups, respectively), H L A - D R + (p = 0 . 0 2 for control group), H L A - D Q + (p = 0.0003 and p = 0.02), and C D 4 + C D 8 + ( p = 0.04 for p o s t - C T group).
-+ 105 -+ 12 +- 37 -+ 140 +_ 104 -+ 44 -+ 108
W e have analysed w h e t h e r differences exist between the values of these s u b p o p u l a t i o n s and the levels of cytotoxicity over Raji in the p o s t - C T patient group. For the analysis, we divided the p o s t - C T group in two subgroups: (A) 10 patients that presented levels lower than or equivalent to 40% of lysis over Raji cell line at E : D of 80 : 1; and (B) 19 patients that surpass 40% of lysis. In g r o u p B, the percentages and absolute counts of the C D 2 5 + (p = 0.02 and p = 0.02, respectively), C D 6 9 + (p = 0.03 and p = 0.02) and HLA-DR + (p = 0.04 and p = 0.001) subpopulations were significantly increased in relation to g r o u p A (Fig. 5). Discussion In our study, the percentages of cytolytic activity of the P B M C s not stimulated by IL-2 o v e r Raji cells (spontaneous L A K activity) were correlated with the percentages of the l y m p h o c y t e s u b p o p u l a t i o n s with p h e n o t y p e C D 5 6 +, C D 3 C D 8 +, C D 3 - C D 5 6 + and CD3 C D 5 7 + in patients with acute l e u k e m i a in C R , both p o s t - C T and p o s t - A B M T . In the p o s t - A B M T group, it was also correlated with the p e r c e n t a g e s of the C D 3 C D 1 6 + subpopulation. In the control group, the subpopulations C D 3 C D 8 + and C D 3 - C D 5 6 + were correlated with s p o n t a n e o u s L A K activity with a significance of p < 0.1. W e have observed that these s u b p o p u l a t i o n s were basically the same as those that correlate with the N K activity
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PATIENTS GROUP A
PATIENTS GROUP B
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Fig. 5. Comparison between the percentage of lymphocyte subpopulations with CD25+, CD69+ and HLA-DR+ before (PBL) and after 3 day culture with IL-2 (LAK). Group A: post*CT patients who had <~40% for lysis of Raji cell line at E:T ratio of 80: 1. Group B: post-CT patients who had >40% for lysis of Raji cell line at E:T ratio of 80: 1. Results are means -+ S.D.
measured over the K562 cell line [20]. These results suggest that the subpopulations themselves are responsible for the NK activity and for the spontaneous LAK activity. The absence of a good correlation between spontaneous LAK activity and the lymphocyte subpopulations in the control group, contrary to what occurs with NK activity [20], may have been caused by the resistance of the Raji cells to lysis by the PBMCs in most of the normal individuals. On the other hand, there were some patients in the post-CT and post-ABMT groups that showed an increased spontaneous LAK activity (16.1% of post-CT and 24.5% of post-ABMT patients overcame confidence limits for spontaneous lysis over Raji at E : T of 80 : 1). These patients also presented the highest percentages among the aforementioned subpopulations and, as a result, may have contributed to the significance of
the correlation between both parameters in both groups of patients. The equivalence between the subpopulations responsible for the NK activity and the spontaneous LAK activity may suggest that faced with the K562 and the Raji cell line the same lytic mechanisms are developed although with different degrees of efficiency. This hypothesis is supported by data previously obtained in our laboratory [8] showing that the percentages of lyric activity over both cell lines are correlated between the two groups of patients and among normal individuals. These correlations existed before and after incubation with IL-2. In cultures of PBMCs with IL-2 we have observed significant increases in the percentages of subpopulations of T origin CD8 +, CD3+CD8 + and CD4+CD8 +. Other authors have observed increases in the T populations CD5 + [2] and CD8 + [21,22]. We have observed a significant absolute increase for CD4+CD8 +, and only moderate absolute increases for CD8 + and CD3+CD8 +. As the short-term culture with IL-2 produces a significant cell loss, as shown by our results and those of other authors [23, 24], the percentage increase of T subpopulations may be explained by their enrichment in culture rather than by their growth. Some investigators have demonstrated via purification of certain lymphocyte subpopulations with AcMo and later culture with IL-2, that the greatest capacity for generation of LAK activity lies in precursors with NK phenotype (NK-LAK) CD3 CD56 + and/or CD16 + [2, 25-27] that may or may not express the CD8 marker. A lower proportion of LAK activity may be generated by subpopulations with T phenotype (T-LAK) CD3+CD16 - [21]. In our report there is a fraction of patients that show an immunocompromise to generate in vitro LAK activity since they do not reach the levels of cytolytic activity obtained by the control group in our laboratory [8] over K562 and/or Raji (>40%) despite the fact that the mean initial percentages of the NK-LAK (CD3-CD16 +, CD3 CD56 +, CD3-CD8 +) precursor subpopulations do not differ significantly from the percentages obtained by the patients of the group with a lytic activity higher than 40%. Purification experiments with the remaining subpopulations of the culture with IL-2 revealed that the effector cells of the LAK activity maintain the initial phenotype of NK or T cells [2, 3, 28]. The more potent LAK effectors are those with a CD3 CD4 CD16 + phenotype although there are also T-LAK effectors [26, 27, 29, 30]. We have not observed growth of the NK-LAK effectors. Nevertheless, the increase in LAK activity in the shortterm culture with IL-2 was correlated with percentage
LAK cytotoxicity and lymphocyte subpopulations and absolute increases in the CD25 +, CD69 + and H L A - D R + subpopulations. It has been proven that the expression of these markers may be induced in both M H C mediated and M H C independent lymphocyte activation [22, 24, 31-34]. Modulation of the m a r k e r CD25 may be due to the induction of the high affinity IL-2 receptor on the lymi3hocyte surface [35-37]. The CD69 and H L A - D R antigens may be involved in the regulation of lymphocyte activation and differentiation. According to our results, those patients with worst induction of the in vitro generated L A K activity are the ones that express these markers at lower percentages after culture with IL-2. Both data reflect the difficulty of the lymphocyte response to IL-2 in these patients. The analysis of the percentages of the spontaneous and of the in vitro generated L A K activity, together with the determination of the percentages and/or the absolute counts of the phenotype markers correlated with them, may contribute to the evaluation of the potential capacity of response to a therapeutic intervention with IL-2 in patients with acute leukemia.
Acknowledgements--This work has been supported by a grant from the Fondo de Investigaciones Sanitarias de la Seguridad Social (FISss no. 91/0384). We gratefully acknowledge E. Cort6s, G. de la Concha and R. Guzmfin for expert technical assistance, and F. Morote for her translation of the manuscript.
References 1. Henney C. S., Kuribayashi K., Kern D. E. & Gillis S. (1981) Interleukin-2 augments natural killer activity. Nature 291,335. 2. Phillips J. H. & Lanier L. L. (1986) Dissection of the lymphokine-activated killer phenomenon. Relative contribution of peripheral blood natural killer cells and T-lymphocytes to cytolysis. J. exp. Med. 164, 814. 3. Ortaldo J. R., Mason A. & Overton R. (1986) Lymphokine-activated killer cells. Analysis of progenitors and effectors. J. exp. Med. 164, 1193. 4. Ballas Z. K. & Rasmussen W. (1990) Lymphokineactivated killer (LAK) cells. IV. Characterization of murine LAK effector subpopulations. J. lmmun. 144, 386. 5. Fuchshuber P. R., Lotzov~i E. & Savary C. A. (1992) Generation of MHC-nonrestricted and restricted oncolytic subsets from human bone marrow. Cell. Immun. 139, 30. 6. Lotze M. T., Grimm E. A., Mazumder A., Strausser J. L. & Rosenberg S. A. (1981) In vitro growth of cytotoxic human lymphocytes. IV. Lysis of fresh and cultured autologous tumor by lymphocytes cultured in T cell growth factor. Cancer Res. 41, 4420. 7. Grimm E. A., Mazumder A., Zhang H. Z. & Rosenberg S. A. (1982) Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous
821
human peripheral blood lymphocytes. J. exp. Med. 155, 1823. 8. Parrado A., Rodriguez-Fernfindez J. M., Casares S., Noguerol P., Plaza E., Parody R., Espigado I., Bias J. M. de & Garcia-Solis D. (1993) Generation of LAK cells in vitro in patients with acute leukemia. Leukemia 7, 1344. 9. Lotzovfi E., Savary C. A. & Herberman R. B. (1987) Induction of NK cell activity against fresh human leukemia in culture with interleukin 2. J. Immun. 138, 2718. 10. Dawson M. M., Johnston D., Taylor G. M. & Moore M. (1986) Lymphokine activated killing of fresh human leukaemias. Leukemia Res. 10, 683. 11. Oshimi K., Oshimi Y., Akutsu M., Takei Y., Saito H., Okada M. & Mizoguchi H. (1986) Cytotoxicity of interleukin 2-activated lymphocytes for leukemia and lymphoma cells. Blood 68, 938. 12. Panayotides P., Porwit A., Sj6gren A. M., Wasserman J. & Reizenstein P. (1988) Resistance of some leukemic blasts to lysis by lymphokine activated killer (LAK) cells. Eur. J. Haemat. 40, 362. 13. Fierro M. T., Liao X. $2, Lusso P., Bonferroni M., Matera L., Cesano A., Lista P., Arione R., Forni G. & Foa R. (1988) In vitro and in vivo susceptibility of human leukemic cells to lymphokine activated killer activity. Leukemia 2, 50. 14. Adler A., Chervenick P. A., Whiteside T. L., Lotzovfi E. & Herberman R. B. (1988) Interleukin 2 induction of lymphokine-activated killer (LAK) activity in the peripheral blood and bone marrow of acute leukemia patients. I. Feasibility of LAK generation in adult patients with active disease and in remission. Blood 71, 709. 15. Adler A., Albo V., Blatt J., Whiteside T. L. & Herberman R. B. (1989) Interleukin-2 induction of lymphokine-activated killer (LAK) activity in the peripheral blood and bone marrow of acute leukemia patients: II. Feasibility of LAK generation in children with active disease and in remission. Blood 74, 1690. 16. Teichmann J. V., Ludwig W. D., Seibt-Jung H. & Thiel E. (1989) Induction of lymphokine-activated killer cells against human leukemia cells in vitro. Blur 59, 21. 17. Archimbaud E., Bailly M. & Dor6 J. F. (1991) Inducibility of lymphokine activated killer (LAK) cells in patients with acute myelogenous leukaemia in complete remission and its clinical relevance. Br. J. Haemat. 77, 328. 18. Archimbaud E., Thomas X., Campos L., Magaud J. P., Fiere D. & Dor6 J. F. (1991) Susceptibility of adult acute lymphoblastic leukemia blasts to lysis by lymphokine-activated killer cells. Leukemia 5, 967. 19. Findley H. W. Jr, Mageed A. A., Nasr S. A. & Ragab A. H. (1988) Recombinant interleukin-2 activates peripheral blood lymphocytes from children with acute leukemia to kill autologous leukemic cells. Cancer 62, 1928. 20. Parrado A., Casares S. & Rodriguez-Fermindez J. M. (1994) Natural killer cytotoxicity and lymphocyte subpopulations in patients with acute leukemia. Leukemia Res. 18, 191. 21. Sawada H., Abo T., Sugawara S. & Kumagai K. (1988) Prerequisite for the induction of lymphokine-activated killer cells from T lymphocytes. J. lmmun. 140, 3668. 22. Higuchi C. M., Thompson J. A., Cox T., Lindgren C. G., Buckner C. D. & Fefer A. (1989) Lymphokineactivated killer function following autologous bone
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A. Parrado et al.
marrow transplantation for refractory hematological malignancies. Cancer Res. 49, 5509. 23. Boldt D. H., Mills B. J., Gemlo B. T., Holden H., Mier J., Paietta E., McMannis J. D., Escobedo L. V., Sniecinski I., Rayner A. A., Hawkins M. J., Atkins M. B., Ciobanu N. & Ellis T. M. (1988) Laboratory correlates of adoptive immunotherapy with recombinant interleukin-2 and lymphokine-activated killer cells in humans. Cancer Res. 48, 4409. 24. Kubo K., Tachino J., Ichikawa J., Hamamoto K. & Ueda K. (1991) HLA typing using IL-2 activated T lymphocytes: usefulness in pediatric candidates for allogeneic bone marrow transplantation. Bone Marrow Transpl. 8, 185. 25. Itoh K., Tilden A. B., Kumagai K. & Balch C. M. (1985) Leu-ll + lymphocytes with natural killer (NK) activity are precursors of recombinant interleukin 2induced activated killer (AK) cells. J. Immun. 134, 802. 26. Tilden A. B., Itoh K. & Balch C. M. (1987) Human lymphokine-activated killer (LAK) cells: identification of two types of effector cells. J. Immun. 138, 1068. 27. Ferrini S., Miescher S., Zocchi M. R., Fliedner V. v o n & Moretta A. (1987) Phenotypic and functional characterization of recombinant interleukin 2 (rIL-2)induced activated killer cells: analysis at the population and clonal levels. J. Immun. 138, 1297. 28. Lanier L. L., Le A. M., Civin C. I., Loken M. R. & Phillips J. H. (1986) The relationship of CD16 (Leu11) and Leu-19 (NKH-1) antigen on human peripheral blood NK cells and cytotoxic T lymphocytes. J. Immun. 136, 4480. 29. Grimm E. A., Ramsey K. M., Mazumder A., Wilson D. J., Djeu J. Y. & Rosenberg S. A. (1983) Lymphokine-activated killer cell phenomenon. II. Precursor phenotype is serologically distinct from
peripheral T lymphocytes, memory cytotoxic thymusderived lymphocytes, and natural killer cells. J. exp. Med. 157, 884. 30. Kalland T., Belfrage H., Bhiladvala P. & Hedlund G. (1987) Analysis of the murine lymphokine-activated killer (LAK) cell phenomenon: dissection of effectors and progenitors into NK- and T-like cells. J. Immun. 138, 3640. 31. London L., Perussia B. & Trinchieri G. (1985) Induction of proliferation in vitro of resting human natural killer cells: expression of surface activation antigens. J. Immun. 134, 718. 32. Lanier L. L., Buck D. W., Rhodes L., Ding A., Evans E., Barney C. & Phillips J. H. (1988) Interleukin 2 activation of natural killer cells rapidly induces the expression and phosphorylation of the Leu-23 antigen. J. exp. Med. 167, 1572. 33. Letellier C., Rameliarison L., Fizet D., Ferrer A. M. & Vezon G. (1991) The influence of cryopreservation on activity and surface markers of lymphokine-activated killer cells. Vox Sang 61, 90. 34. Baume D. M., Robertson M. J., Levine H., Manley T. J., Schow P. W. & Ritz J. (1992) Differential responses to interleukin 2 define functionally distinct subsets of human natural killer cells. Eur. J. Immun. 22, 1. 35. Siegel J. P., Sharon M., Smith P. L. & Leonard W. J. (1987) The IL-2 receptor fl chain (pT0): role in mediating signals for LAK, NK, and proliferative activities. Science 238, 75. 36. Kehrl J. H., Dukovich M., Whalen G., Katz P., Fauci A. S. & Greene W. C. (1988) Novel interleukin 2 (IL2) receptor appears to mediate IL-2-induced activation of natural killer cells. J. clin. Invest. 81,200. 37. Owen-Schaub L. B., Loudon W. G., Yagita M. & Grimm E. A. (1988) Functional differentiation of human lymphokine-activated killing (LAK) is distinct from expansion and involves dissimilar interleukin 2 receptors. Cell. Immun. 111, 235.
Leukemia Research Vol. 18, No. 11, pp. 815-822, 1994 Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0145 2126/94 $7.00 + 0.00
0145-2126(94)00093-X LYMPHOKINE-ACTIVATED KILLER CYTOTOXICITY AND LYMPHOCYTE SUBPOPULATIONS IN PATIENTS WITH ACUTE LEUKEMIA Antonio Parrado, Sofia Casares and Juan-Manuel Rodriguez-Fernfindez Servicio de Hematologla y Hemoterapia, Hospital Universitario Virgen del Rocio, Servicio Andaluz de Salud, Sevilla, Spain (Received 15 February 1994. Revision accepted 18 July 1994) Abstract--In this report we investigated lymphokine-activated killer (LAK) cytotoxicity and lymphocyte subpopulations of peripheral blood mononuclear cells (PBMCs) of patients with acute leukemia in complete remission after chemotherapy or autologous bone marrow transplantation (ABMT) and of normal donors. A positive linear correlation was found between the percentage of spontaneous LAK activity and that of lymphocyte subpopulations with phenotypes CD56 +, CD3-CD8 + CD3-CD56 ÷ and CD3 CD57 ÷ in both groups of patients. A 3day culture with IL-2 produced an up-regulation in the expression of the CD25, CD69, and HLADR markers proportional to the LAK cytotoxicity levels generated in the culture. Determination of percentages of spontaneous and in vitro generated LAK activity as well as of the abovementioned phenotypic markers contribute to the analysis of the process of LAK activation in patients with acute leukemia and may also be useful in those cases in which immunotherapy with IL-2 and/or LAK cells is anticipated. Key words: LAK cells, acute leukemia, chemotherapy, ABMT, cytotoxicity, flow cytometry.
the NK-resistant targets by MC without incubation with IL-2 is termed spontaneous LAK activity. In vitro generated LAK cells in patients with acute leukemia and in controls are able to lyse to a certain degree the allogeneic and autologous leukemic blasts [8-19], thus supporting the use of IL-2 and/or LAK cells as treatment against residual minimal leukemia. We have previously observed that the percentages of NK activity developed by peripheral blood mononuclear cells (PBMC) over the K562 cell line are related to the percentages of lymphocyte population with CD3-CD16 +, CD3 CD56 + and CD3-CD8 + phenotypes in patients with acute leukemia in complete remission (CR) and in normal individuals [20]. In this study we found correlations between the percentages of some lymphocyte subpopulations defined by monoclonal antibodies (MoAb) and the percentages of spontaneous LAK activity measured over the Raji cell line in the PBMCs of patients with acute leukemia in CR after chemotherapy (CT) and autologous bone marrow transplantation (ABMT). Moreover, we have determined how some phenotypic markers are modulated in the in vitro generation of LAK activity.
Introduction Lymphokine-activated killer (LAK) activity is a form of cytotoxicity generated from non-preimmunized mononuclear cells (MC) when submitted to shortterm culture with IL-2 [1]. Most LAK cells seem to arise from natural killer (NK) cells, although T-cells may also play a role [2-5]. Recognition and lysis mechanisms developed by NK and LAK cells do not imply the involvement of the major histocompatibility complex (MHC). LAK activity appears when using autologous or allogeneic target tumor cells resistant to the NK activity [6, 7]. The cytotoxic activity exerted over Abbreviations: LAK, lymphokine-activated killer; PBMC, peripheral blood mononuclear cells; ABMT, autologous bone marrow transplantation; 1L-2, interleukin 2; MC, mononuclear cells; NK, natural killer; MHC, major histocompatibility complex; MoAb, monoclonal antibodies; CR, complete remission; CT, chemotherapy; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; FITC, fluorescein-isothiocyanate; PE, phycoerythrin; E: T, effector to target ratio; CI, confidence interval; PBL, peripheral blood lymphocytes. Correspondence to: Dr Antonio Parrado, Servicio de Hematologia y Hemoterapia, Hospital Universitario Virgen del Rocio, Avda. Manuel Siurot, s/n, 41013 Sevilla, Spain (Fax: (5)4248124).
815
A+ Parrado et al.
816
Patients
80
Materials and Methods
70
We studied 31 patients with acute leukemia in CR postCT, including 14 cases with acute lymphoblastic leukemia (ALL) and 17 with acute myeloid leukemia (AML); 53 patients with acute leukemia post-ABMT, including 29 ALL and 24 AML. Fourteen normal donors served as a control group. PBMCs were obtained by standard Ficoll density gradient centrifugation from normal donors, from post-CT patients 0-27 months (median 8 months) after the end of the induction therapy and from post-ABMT patients 0-43 months (median 9 months) after ABMT.
Generation of LA K cells PBMCs from 29 patients in the post-CT group (13 ALL and 16 AML) and from nine normal donors were cultured for 3 days at a concentration of 10 6 cells/ml in RPMI 1640 medium with 5% human AB serum (complete medium) and 500U/ml natural IL-2 obtained from human lymphocyte cultures (Biotest Pharma GmbH, Dreieich, Germany). PBMCs cultured in parallel for 3 days in complete medium without the addition of IL-2 served as controls.
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TCR~,6/CD8, CD3/CD16, CD4/CD16, CDS/CD16,
TCRoLfl/CDI6, TCRy6/CD16, CD3/CD56, CD4/CD56, CD8/CD56, TCRo,fl/CD56, TCRy6/CD56, CD57/CD3, CD57/CD4, CD57/CD8, CD16/CD56, CD57/CD16, CD57/CD56. Lymphocytes were gated using forward and side light scatter in combination with CD45/CD14 immunofluorescence.
Cytotoxicity assay The human lymphoid Raji was used as a target to measure LAK activity as previously described [8]. Briefly, 5 X 10 6 Raji cells were labeled by incubation with 100 ~tCi 51Cr for 90 rain. In triplicate, 5 x 103 target (T) cells and varying numbers of effector (E) cells at E : T ratios of 2.5 : 1, 10:1, 40:1 and 80:1 suspended in RPMI-1640 medium containing 5% heat-inactivated human AB serum were incubated together in 96-well round-bottomed microtiter plates for 4 h. Control wells containing target cells alone in the medium or 2% sodium dodecyl sulphate (SDS) were used to measure spontaneous release (SR) and maximum release (MR) of chromium. The supernatants were collected using the Skatron Supernatant Collection System and measured in a gamma counter. The percentage of experimental lysis was calculated according to the following formula: % lysis = 100 x (ER - SR)/(MR - SR), where ER represents experimental release of SaCr.
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Lymphocyte subpopulations Surface marker analysis was performed in a FACScan which utilized the CONSORT 32 system and the software application LYSYS II (Becton-Dickinson, San Jose, CA) using the following MoAb: anti-CD2 (Leu-5b), CD3 (Leu4), CD4 (Leu-3), CD8 (Leu-2), CD16 (Leu-ll), CD19 (Leu-12), CD20 (Leu-16), CD25, CD56 (Leu-19), CD57 (Leu-7), CD69 (Leu-23), HLA-DR, HLA-DQ, TCRoLfl, TCRy6 (Becton Dickinson, Mountain View, CA) and polyclonal anti-kappa and anti-lambda antibodies (Dakopatts, Glostrup, Denmark). We performed double labeling with fluorescein-isothiocyanate (FITC) and phycoerythrin (PE) conjugated MoAb in the following combinations: CD2/ CD3, CD3/CD4, CD3/CD8, CD4/CD8, TCRo:fl/CD3, TCRafl/CD4, TCRolfl/CD8, TCR76/CD3, TCRy6/CD4,
,
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Fig. 1. Correlation of spontaneous LAK activity and lymphocyte subpopulation CD56 + for: (A) post-CT; and (B) post-ABMT patients. Spontaneous LAK activity was measured against Raji cell line at E : T ratio of 80:1; r represents Pearson's correlation coefficient; p is the significance of r; a low p-value implies that the data have a significant linear trend.
Statistical analysis" Correlations were calculated with the simple linear regression analysis. Differences between groups were confirmed using Student's t-test. Two-tailed p values of 0.05 or less were considered significant. Results
Relationship between spontaneous L A K activity and lymphocyte subpopulations Significant linear correlations were found between the percentages of cytolytic activity of the PBMCs over the Raji cell line and the percentages of lymphocyte subpopulations with phenotype CD56 + (Fig. 1), C D 3 - C D 8 + (Fig. 2), C D 3 - C D 5 6 + (Fig. 3) and C D 3 - C D 5 7 + (Fig. 4) in both patient groups (postCT and post-ABMT). A m o n g the controls, the correlations between spontaneous L A K activity and subpopulations C D 3 - C D 8 + (r = 0.531, p = 0.05) and CD3 CD56 + (r = 0.469, p = 0.09) were close to the level of significance. Correlations between the spontaneous L A K activity and the subpopulations CD3 C D I 6 ÷ (r = 0.497, p = 0.000), CD16+CD56 + (r = 0.358, p = 0.009), CD16+CD57 + (r = 0.367, p = 0.007) and CD56+CD57 + (r = 0.423, p = 0.002) were only found for the p o s t - A B M T group. Mean percentages of spontaneous L A K activity were 5.6-+ 7.5 and 8.2-+ 9.9 for the post-CT group at
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Fig. 4. Correlation of spontaneous LAK activity and lymphocyte subpopulation CD3-CD57 + for: (A) post-CT; and (B) post-ABMT patients. Spontaneous LAK activity was measured against Raji cell line at E : T ratio of 80 : 1.
Relationship between in vitro generated LAK activity and lymphocyte subpopulations
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the control group, respectively. At E : T of 80 : 1,5 out of 31 (16.1%) post-CT patients overcame confidence limits at 95% for spontaneous L A K activity (Confidence interval (CI), 4.7-11.6%), as well as 13 out of 53 (24.5%) post-ABMT patients (CI, 6.2-11.9%), and 1 out of 14 (7.1%) controls (CI, 0.6-11.7%).
r=0.614
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Fig. 2. Correlation of spontaneous LAK activity and lymphocyte subpopulation CD3-CD8 + for: (A) post-CT; and (B) post-ABMT patients. Spontaneous LAK activity was measured against Raji cell line at E : T ratio of 80 : 1.
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Fig. 3. Correlation of spontaneous LAK activity and lymphocyte subpopulation CD3 CD56 + for: (A) post-CT; and (B) post-ABMT patients. Spontaneous LAK activity was measured against Raji cell line at E : T ratio of 80 : 1.
E : T of 40:1 and 80:1, respectively, 6.4 + 8.4 and 9 . 1 + 10.5 for the p o s t - A B M T at the same E : T ratios, respectively, and 4.6 _+ 6.5 and 6.1 _+ 9.6 for
Mean percentages of lytic activity over the Raji cell line and of the different lymphocyte subpopulations before and after culture with and without IL-2 are shown in Tables 1 and 2. Initial percentages of lysis were not correlated with those obtained after incubation with IL-2. Significant percentage increases were observed in the cultures without IL-2 in relation to the initial values for the subpopulations CD3 + (p = 0.0005), CD4 + (p = 0.0006) and TCRcq3 + (p = 0.02) in the post-CT group and for CD3 + (p = 0.008), CD4 + (p = 0.0003), TCRcrfl + ( p = 0 . 0 1 ) , CD69 + ( p = 0.04), H L A - D R + (p = 0.01) and C D 3 + C D 8 + (p = 0.008) in the control group. Significant percentage decreases were observed in these cultures for the subpopulations CD16 + (p = 0.05), H L A - D R + (p = 0.004), C D 3 - C D 1 6 + (p = 0.003), C D 3 - C D 5 6 + (p = 0.002) and CD16+CD56 + (p = 0.01) in the post-CT group and for CD3-CD8* (p = 0.0009),
818
A. Parrado et al. Table 1. P e r c e n t a g e (mean_+ S.D.) of lymphokine-activated killer activity in p o s t - C T p a t i e n t s and n o r m a l d o n o r s b e f o r e (day 0) and after culture (day 3) without or with IL-2 P o s t - C T patients
Normal donors
E:T
- I L - 2 (0)
- I L - 2 (3)
+ I L - 2 (3)
- I L - 2 (0)
- I L - 2 (3)
+ I L - 2 (3)
2.5 : 1 10:1 40:1 80:1
2.5 -+ 3.0 3.2 -+ 4.8 5.9_+7.7 8.5+10.1
0.7 + 1.3 1.5 -+ 2.1 2.6_+3.1 4.1_+4.1
10.4 -+ 14.3 22.0 _+ 20.4 35.7-+25.6 43.5-+25.5
1.4 + 1.7 1.2 + 1.7 3.0-+2.2 3.9-+3.1
0.4 -+ 1.3 0.6 -+ 1.7 1.6_+4.3 2.6-+6.6
8.8 -+ 4.2 24.7 -+ 8.8 47.1_+11.9 54.2-+8.5
Table 2. P e r c e n t a g e ( m e a n -+- S . D . ) of l y m p h o c y t e subpopulations in p o s t - C T patients and normal d o n o r s b e f o r e (day 0) and after culture (day 3) without or with IL-2 Post-CT patients - I L - 2 (0) CD3 + CD4 + CD8 + CD4+CD8 + CD4+/CD8 + TCRa~fl + TCRy6 + CD2 + CD16 + CD56 + CD57 + CD25 + CD69 + HLA-DR + HLA-DQ + CD19 + CD20 + SIgK÷ sIg;. + CD3+CD8 + CD3-CD8 + CD3+CD16 + CD3-CD16 + CD4+CD16 + CD8+CD16 + CD3+CD56 + CD3-CD56 + CD4+CD56 + CD8+CD56 + CD3+CD57 ÷ CD3-CD57 + CD4+CD57 + CD8+CD57 + CD16+CD56 + CD16+CD57 + CD56+CD57 +
74.8 35.1 31.0 2.4 1.44 67.1 8.8 84.5 21.7 31.8 24.3 7.3 17.7 26.1 6.4 6.4 6.6 2.9 1.5 26.0 4.9 11.7 10.1 2.9 7.4 18.9 12.8 5.2 11.5 18.6 5.7 3.4 13.0 14.5 9.1 11.3
-+ 13.9 _+ 12.6 _ 12.5 _+ 2.8 _+ 1.18 + 15.3 + 7.9 -+ 10.3 -+ 11.6 +- 12.2 + 15.3 + 3.2 -+ 10.8 -+ 13.9 -+ 4.6 + 6.0 -+ 5.0 --+ 3.5 --+ 2.2 -+ 12.9 + 4.6 _+ 8.8 -+ 7.8 -+ 2.4 -+ 8.0 -+ 9.0 -+ 9.0 -+ 4.9 -+ 7.9 -+ 14.3 + 5.4 -+ 2.7 +- 9.9 -+ 10.4 -+ 8.8 +- 8.4
C D 3 C D 1 6 + ( p = 0.03) and C D 3 in t h e c o n t r o l g r o u p .
- I L - 2 (3) 87.5 48.3 32.7 2.6 1.94 77.2 10.1 88.7 15.1 24.6 19.8 6.6 16.1 19.7 5.7 3.5 5.0 1.1 0.6 29.5 3.2 10.9 4.6 4.1 5.5 17.8 5.4 6.5 10.8 16.4 3.4 4.0 11.2 8.4 5.6 7.6
-+ 10.5 _+ 15.5 _+ 13.4 _+ 3.0 _+ 1.59 + 13.5 -+ 9.7 -+ 11.1 +- 9.9 -+ 11.1 +- 12.8 -+ 4.2 +- 8.8 +- 10.0 -+ 4.0 -+ 3.9 -+ 3.9 --+ 1.7 --+ 1.1 -+ 13.6 -+ 4.3 -+ 9.1 -+ 3.8 -+ 2.8 -+ 3.9 -+ 8.3 -+ 5.1 -+ 3.2 + 7.3 -+ 12.6 -+ 4.4 -+ 2.8 -+ 8.7 -+ 5.0 +- 7.2 + 5.2
C D 5 6 + ( p = 0.01)
S i g n i f i c a n t p e r c e n t a g e i n c r e a s e s w e r e o b s e r v e d in t h e c u l t u r e s w i t h I L - 2 in r e l a t i o n t o t h e initial v a l u e s
Normal d o n o r s + I L - 2 (3) 87.3 _+ 10.5 45.1 _+ 14.9 41.5 _+ 14.3 5.7 _+ 4.9 1.38 _+ 1.13 78.1 -+ 11.3 12.3 + 6.3 93.2 +- 5.3 17.1 +- 8.8 30.9 -+ 13.3 26.1 -+ 13.3 16.8 -+ 11.8 38.4 -+ 19.2 39.3 -+ 16.5 16.3 -+ 11.5 3.9 + 2.9 4.8 -+ 3.9 1.2--+ 1.5 0.6 --+ 0.9 37.1 -+ 14.9 4.2 +- 4.1 12.0 + 7.2 5.1 -+ 5.4 4.1 -+ 2.9 7.3 + 4.6 22.7 -+ 12.2 8.3 -+ 7.2 8.4 -+ 5.4 15.3 -+ 10.1 21.9 -+ 12.4 4.2 +- 5.1 6.0 -+ 4.6 16.9 -+ 11.9 12.4 -+ 7.5 7.7 -+ 6.0 11.6 -+ 8.4
- I L - 2 (0) 72.6 40.2 29.8 1.1 1.40 66.4 3.4 82.1 19.4 27.1 23.1 7.7 10.6 17.6 6.1 7.2 7.1 3.1 2.1 22.0 7.8 6.4 13.0 1.7 7.3 13.1 14.0 2.9 10.8 15.0 8.1 2.6 15.1 15.2 9.9 11.8
_+ 9.4 _+ 4.1 _+ 7.8 _+ 2.1 _+ 0.25 -+ 9.1 + 2.5 -+ 8.0 + 5.6 -+ 5.4 -+ 9.4 -+ 1.8 -+ 7.6 + 7.8 -+ 3.7 -+ 3.7 -+ 2.2 --+ 2.0 --+ 1.3 -+ 8.5 +- 1.8 _+ 5.2 -+ 5.1 _+ 1.8 _+ 1.5 -+ 7.5 _+ 5.5 -+ 2.3 -+ 3.5 _+ 10.5 -+ 4.8 -+ 1.7 -+ 8.9 -+ 5.5 -+ 4.7 -+ 4.1
- I L - 2 (3) 86.4 49.3 36.1 2.0 1.41 79.7 5.2 88.8 18.3 29.1 21.7 7.4 21.1 28.2 7.7 5.6 6.8 1.7 1.1 32.7 3.4 11.6 6.8 4.1 6.9 22.8 6.3 8.9 12.0 16.1 5.6 3.1 16.2 13.7 8.3 10.1
_+ 10.2 + 4.4 _+ 6.5 _+ 1.5 _+ 0.29 -+ 10.6 -+ 3.1 -+ 7.0 -+ 7.6 -+ 11.6 + 9.3 +- 3.1 -+ 12.2 -+ 7.6 -+ 3.6 -+ 3.1 -+ 2.8 _+ 1.7 + 0.9 -+ 6.2 +- 2.6 -+ 9.2 -+ 5.6 -+ 3.1 -+ 2.5 -+ 14.9 -+ 5.6 -+ 7.4 +- 3.3 -+ 9.1 -+ 4.7 -+ 1.4 + 8.5 +- 5.8 -+ 4.0 +- 3.9
+ I L - 2 (3) 86.1 _+ 5.1 51.6 _+ 7.4 39.8 -+ 8.7 3.4 _+ 2.6 1.37 + 0.41 78.7 -+ 6.0 5.6 -+ 2.5 89.6 -+ 4.8 19.4 -+ 8.2 29.4 + 10.2 22.8 -+ 10.7 13.3 +- 4.6 38.9 +- 13.6 43.7 +- 16.5 13.8 -+ 4.9 6.(1 -+ 2.7 6.3 -+ 2.9 1.9 +-- 1.9 1.8 _+ 1.4 34.9 -+ 7.0 4.9 _+ 2.7 12.6 -+ 7.2 6.9 -+ 2.6 4.6 -+ 3.1 8.2 -+ 4.1 21.8 -+ 10.7 7.7 -+ 3.5 8.1 -+ 5.6 13.8 -+ 4.5 18.2 -+ 11.4 4.6 -+ 1.3 5.0 +- 2.5 18.3 -+ 11.1 13.1 -+ 5.6 7.9 -+ 3.3 11.2 -+ 3.0
b o t h in p o s t - C T a n d c o n t r o l g r o u p s f o r t h e s u b p o p u l a t i o n s C D 3 + ( p = 0.0003 a n d p - - 0.0000, respectively), C D 4 + ( p = 0.007 a n d p = 0.0000), C D 8 + ( p = 0 . 0 0 4 a n d p = 0 . 0 0 0 1 ) , TCRa~fi + ( p =
LAK cytotoxicity and lymphocyte subpopulations
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Table 3. PBMC and PBL counts (× 10 ~ ml) and percentage of PBL (mean -+ S.D.) in post-CT patients and normal donors before (day 0) and after culture (day 3) without or with IL-2 Normal donors
Post-CT patients
PBMC counts PBL percentages PBL counts
- I L - 2 (0)
- I L - 2 (3)
+IL-2 (3)
1000 -+ 0 70.8 -+ 17.8 708 -+ 178
632 -+ 205 85.9 +- 16.4 535 -+ 194
643 +- 233 92.2 +- 11.0 594 -+ 227
- I k - 2 (0)
- I L - 2 (3)
+IL-2 (3)
1000 -+ 0 81.6 +- 7.5 816 +- 75
848 +- 277 87.8 +- 6.5 756 -+ 262
770 +- 287 96.7 +- 6.4 752 +- 305
Table 4. Absolute counts (x 10 -3 ml, mean +- S.D.) of lymphocyte subpopulations in post-CT patients and normal donors before (day 0) and after culture (day 3) without or with IL-2 Normal donors
Post-CT patients
CD8 + CD4+CD8 + CD25 + CD69 + HLA-DR + HLA-DQ + CD3+CD8 *
- I L - 2 (0)
- I L - 2 (3)
+IL-2 (3)
- I L - 2 (0)
- I L - 2 (3)
+IL-2 (3)
222 -+ 112 17 _+ 24 50 -+ 22 126 + 88 181 -+ 105 43 -+ 30 188 -+ 112
175 + 103 13 -+ 16 33 -+ 21 87 _+ 58 103 + 71 32 -+ 30 159 _+ 103
244 +- 139 32 -+ 30 101 -+ 88 225 + 162 220 -+ 108 89 -+ 58 221 +- 140
242 +_ 69 9 +- 17 63 -+ 18 86 +- 63 145 _+ 68 51 +- 34 179 +- 75
258 13 57 180 211 63 237
287 +- 131 22 _+ 16 101 +_ 58 301 _+ 191 334 _+ 221 109 +- 62 253 -+ 112
0.003 and p = 0.004), C D 2 + (p = 0.0002 and p = 0.03), C D 2 5 + (p = 0.0001 and p = 0.004), C D 6 9 + (p = 0.0000 and p = 0.0001), H L A - D R + (p = 0.003 and p = 0.0006), H L A - D Q + ( p = 0.0000 and p = 0.002), C D 3 + C D 8 + (p = 0.005 and p = 0.003) and C D 4 + C D 8 + ( p = 0.002 and p = 0.05). Significant p e r c e n t a g e decreases were o b s e r v e d in these cultures for the subpopulations CD16 + (p = 0.04), C D 3 - C D 1 6 + (p = 0.009) and C D 1 6 + C D 5 6 + (p = 0.008) in the p o s t - C T g r o u p and for C D 3 - C D 8 + (p = 0.02), C D 3 - C D 1 6 + ( p = 0.005) and C D 3 C D 5 6 + (p = 0.01) in the control group. A joint analysis of these results shows the existence of p e r c e n t a g e increases in the subpopulations C D 8 +, C D 2 5 +, C D 6 9 +, H L A - D R +, H L A - D Q +, C D 3 + C D 8 ÷ and C D 4 + C D 8 + in cultures with IL-2 in relation to the initial values and those o b t a i n e d after culture w i t h o u t IL-2. A f t e r 3 days of culture without and with IL-2, cell counts suffered significant decreases. L y m p h o c y t e s were enriched in culture, and absolute l y m p h o c y t e counts suffered less p r o n o u n c e d decreases (Table 3). A b s o l u t e counts of the relevant l y m p h o c y t e subpopulations are s h o w n in Table 4. Significant increases in culture with IL-2 were observed for C D 2 5 + ( p = 0.005 for p o s t - C T g r o u p ) , C D 6 9 + (p = 0.0000 a n d p = 0.005 for p o s t - C T and control groups, respectively), H L A - D R + (p = 0 . 0 2 for control group), H L A - D Q + (p = 0.0003 and p = 0.02), and C D 4 + C D 8 + ( p = 0.04 for p o s t - C T group).
-+ 105 -+ 12 +- 37 -+ 140 +_ 104 -+ 44 -+ 108
W e have analysed w h e t h e r differences exist between the values of these s u b p o p u l a t i o n s and the levels of cytotoxicity over Raji in the p o s t - C T patient group. For the analysis, we divided the p o s t - C T group in two subgroups: (A) 10 patients that presented levels lower than or equivalent to 40% of lysis over Raji cell line at E : D of 80 : 1; and (B) 19 patients that surpass 40% of lysis. In g r o u p B, the percentages and absolute counts of the C D 2 5 + (p = 0.02 and p = 0.02, respectively), C D 6 9 + (p = 0.03 and p = 0.02) and HLA-DR + (p = 0.04 and p = 0.001) subpopulations were significantly increased in relation to g r o u p A (Fig. 5). Discussion In our study, the percentages of cytolytic activity of the P B M C s not stimulated by IL-2 o v e r Raji cells (spontaneous L A K activity) were correlated with the percentages of the l y m p h o c y t e s u b p o p u l a t i o n s with p h e n o t y p e C D 5 6 +, C D 3 C D 8 +, C D 3 - C D 5 6 + and CD3 C D 5 7 + in patients with acute l e u k e m i a in C R , both p o s t - C T and p o s t - A B M T . In the p o s t - A B M T group, it was also correlated with the p e r c e n t a g e s of the C D 3 C D 1 6 + subpopulation. In the control group, the subpopulations C D 3 C D 8 + and C D 3 - C D 5 6 + were correlated with s p o n t a n e o u s L A K activity with a significance of p < 0.1. W e have observed that these s u b p o p u l a t i o n s were basically the same as those that correlate with the N K activity
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PATIENTS GROUP B
NORMAL DONORS
Fig. 5. Comparison between the percentage of lymphocyte subpopulations with CD25+, CD69+ and HLA-DR+ before (PBL) and after 3 day culture with IL-2 (LAK). Group A: post*CT patients who had <~40% for lysis of Raji cell line at E:T ratio of 80: 1. Group B: post-CT patients who had >40% for lysis of Raji cell line at E:T ratio of 80: 1. Results are means -+ S.D.
measured over the K562 cell line [20]. These results suggest that the subpopulations themselves are responsible for the NK activity and for the spontaneous LAK activity. The absence of a good correlation between spontaneous LAK activity and the lymphocyte subpopulations in the control group, contrary to what occurs with NK activity [20], may have been caused by the resistance of the Raji cells to lysis by the PBMCs in most of the normal individuals. On the other hand, there were some patients in the post-CT and post-ABMT groups that showed an increased spontaneous LAK activity (16.1% of post-CT and 24.5% of post-ABMT patients overcame confidence limits for spontaneous lysis over Raji at E : T of 80 : 1). These patients also presented the highest percentages among the aforementioned subpopulations and, as a result, may have contributed to the significance of
the correlation between both parameters in both groups of patients. The equivalence between the subpopulations responsible for the NK activity and the spontaneous LAK activity may suggest that faced with the K562 and the Raji cell line the same lytic mechanisms are developed although with different degrees of efficiency. This hypothesis is supported by data previously obtained in our laboratory [8] showing that the percentages of lyric activity over both cell lines are correlated between the two groups of patients and among normal individuals. These correlations existed before and after incubation with IL-2. In cultures of PBMCs with IL-2 we have observed significant increases in the percentages of subpopulations of T origin CD8 +, CD3+CD8 + and CD4+CD8 +. Other authors have observed increases in the T populations CD5 + [2] and CD8 + [21,22]. We have observed a significant absolute increase for CD4+CD8 +, and only moderate absolute increases for CD8 + and CD3+CD8 +. As the short-term culture with IL-2 produces a significant cell loss, as shown by our results and those of other authors [23, 24], the percentage increase of T subpopulations may be explained by their enrichment in culture rather than by their growth. Some investigators have demonstrated via purification of certain lymphocyte subpopulations with AcMo and later culture with IL-2, that the greatest capacity for generation of LAK activity lies in precursors with NK phenotype (NK-LAK) CD3 CD56 + and/or CD16 + [2, 25-27] that may or may not express the CD8 marker. A lower proportion of LAK activity may be generated by subpopulations with T phenotype (T-LAK) CD3+CD16 - [21]. In our report there is a fraction of patients that show an immunocompromise to generate in vitro LAK activity since they do not reach the levels of cytolytic activity obtained by the control group in our laboratory [8] over K562 and/or Raji (>40%) despite the fact that the mean initial percentages of the NK-LAK (CD3-CD16 +, CD3 CD56 +, CD3-CD8 +) precursor subpopulations do not differ significantly from the percentages obtained by the patients of the group with a lytic activity higher than 40%. Purification experiments with the remaining subpopulations of the culture with IL-2 revealed that the effector cells of the LAK activity maintain the initial phenotype of NK or T cells [2, 3, 28]. The more potent LAK effectors are those with a CD3 CD4 CD16 + phenotype although there are also T-LAK effectors [26, 27, 29, 30]. We have not observed growth of the NK-LAK effectors. Nevertheless, the increase in LAK activity in the shortterm culture with IL-2 was correlated with percentage
LAK cytotoxicity and lymphocyte subpopulations and absolute increases in the CD25 +, CD69 + and H L A - D R + subpopulations. It has been proven that the expression of these markers may be induced in both M H C mediated and M H C independent lymphocyte activation [22, 24, 31-34]. Modulation of the m a r k e r CD25 may be due to the induction of the high affinity IL-2 receptor on the lymi3hocyte surface [35-37]. The CD69 and H L A - D R antigens may be involved in the regulation of lymphocyte activation and differentiation. According to our results, those patients with worst induction of the in vitro generated L A K activity are the ones that express these markers at lower percentages after culture with IL-2. Both data reflect the difficulty of the lymphocyte response to IL-2 in these patients. The analysis of the percentages of the spontaneous and of the in vitro generated L A K activity, together with the determination of the percentages and/or the absolute counts of the phenotype markers correlated with them, may contribute to the evaluation of the potential capacity of response to a therapeutic intervention with IL-2 in patients with acute leukemia.
Acknowledgements--This work has been supported by a grant from the Fondo de Investigaciones Sanitarias de la Seguridad Social (FISss no. 91/0384). We gratefully acknowledge E. Cort6s, G. de la Concha and R. Guzmfin for expert technical assistance, and F. Morote for her translation of the manuscript.
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