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Induction of cell surface interleukin 2 receptor alpha chain expression on non-T lymphoid leukemia cells
Pergamon
Leukemia Research Vol. 18, No. 11, pp. 855-859, 1994 Elsevier Science Ltd Printed in Great Britain 0145 2126,/94 $7.00 + 0.00
0145-2126(94)00099-9
INDUCTION OF CELL SURFACE INTERLEUKIN 2 RECEPTOR ALPHA CHAIN EXPRESSION ON NON-T LYMPHOID LEUKEMIA CELLS Kazunori Nakase,* Kenkichi Kita,t Shigeru Shirakawa,t Isao Tanaka,$ and Mitsuru Tsudo§ *The Department of Internal Medicine, Yamada Red Cross Hospital, Misono, Japan; tThe Second Department of Internal Medicine, Mie University School of Medicine, Tsu, Japan; SThe Department of Internal Medicine, Suzuka Kaisei Hospital, Suzuka, Japan; and §The Department of Internal Medicine, Kyoto Katsura Hospital, Kyoto, Japan
(Received 22 April 1994. Accepted 2 July 1994) Abstract--We examined nine cases of adult non-T lymphoid leukemia to investigate the cell surface inducibility of interleukin 2 receptor achain (IL-2Ro~) and #chain (IL-2R#) after in vitro culture with and without recombinant human interleukin-l# (rhlL-l#). Induction of IL-2R~was observed in four of six cases with precursor B-cell acute lymphoblastic leukemia (pre-B ALL) and in all of three cases with B-cell mature lymphoid neoplasm (two chronic lymphocytic leukemia and one leukemic phase of non-Hodgkin's lymphoma). All of the IL-2Ro~-inducible cases could express this spontaneously even without rhlL-l#, while IL-2R# did not appear on leukemic cells from any of the cases tested. IL-2Ro~-inducible pre-B ALL cases displayed stern cell antigen CD34 and induced myeloid-associated antigen CD13 simultaneously. These results suggest that IL-2Ro~but not IL-2R# is easily inducible in certain cases of mature B-cell lymphoid neoplasm and pre-B ALL with immature characteristics.
Key words: Interleukin 2 receptor alpha chain, non-T lymphoid leukemia, CD34, CD13.
Introduction
indicate that IL-2Rcr but not IL-2R/3 is inducible in mature B-cell lymphoid disorders and precursor Bcell acute lymphoblastic leukemia with immature properties.
Interleukin 2 (IL-2), initially referred to as a T-cell growth factor [1], must bind to a specific cell surface IL-2 receptor (IL-2R) to play its biological role [2]. The IL-2R is formed by three different peptides (o~, /3 and 7 chains) [3]. The expression of IL-2R is demonstrated not only in T-cell lymphocytes but also in various non-T hematologic disorders [4-8]. However, little is known about the regulation of IL2R expression in non-T leukemia cells. We have recently described the easily induced expression of IL-2Rc~ chain (IL-2Rc 0 on immature acute myelocytic leukemia cells [9]. In the present study, we investigated the cell surface inducibility of two subunits of IL-2R ( a and /3 chains) on non-T lymphoid leukemia cells and analyzed the cellular characteristics of its inducible cases in relation to immunophenotype. Our results
Materials and Methods
Patients and diagnosis Nine adult patients (over 15 years old) with non-T lymphoid leukemia were referred for this study. The diagnoses were as follows; six precursor B-cell acute lymphoblastic leukemia (pre-B ALL) (four CD19 + CD10-ALL and two common ALL), two B-cell chronic lymphocytic leukemia (B-CLL) and one leukemic phase of non-Hodgkin's lymphoma. hnrnunophenotyping Mononuclear cells (MNC) were obtained from bone marrow or peripheral blood by Ficoll-Hypaque density gradient centrifugation. In some cases, the separated MNC were frozen in liquid nitrogen with 20% heat inactivated fetal calf serum (FCS, GIBCO Lab., Chargrin Falls) and 10% dimethylsulfoxide, and thawed immediately before use. In order to prevent non-specific binding of monoclonal antibody (mAb) with receptor for the IgG Fc portion, the samples were pretreated in Hank's medium supplemented with 5% heat inactivated human AB serum. Cells were stained by indirect immunofluorescence, as previously described [10]. CDllb (OKM1), CD13 (MCS2), CD33
Abbreviations: IL-2Rcr, interleukin 2 receptor alpha chain; IL-2R#, interleukin 2 receptor beta chain; ALL, acute lymphoblastic leukemia; CLL, chronic lymphocytic leukemia; IL-I#, interleukin 1#. Correspondence to: Kazunori Nakase, Department of Internal Medicine, Yamada Red Cross Hospital, Misono 810, Mie 516, Japan (Tel: 596-28-2171; Fax 596-28-2965). 855
856
K. Nakase et al. Table 1. Induction of IL-2R on non-T lymphoid leukemia cells IL-1/3 ( - ) Pre-B CD19 (+) CD10 ( - ) ALL Common ALL Mature B CLL LP of lymphoma Total
IL-1/3 (+)
IL-2Ro:
1L-2R/3
IL-2Ro:
IL-2R/3
3/4 1/2
0/3 0/1
2/2 1/2
0/2 0/1
2/2 1/1 7/9
0/2 NT 0/6
2/2 1/1 6/7
0/2 NT 0/5
Number of IL-2R inducible cases / number of cases tested. LP of lymphoma, leukemic phase of non-Hodgkin's lymphoma; NT, not tested.
(My9) and CD34 (My10) were tested as myeloid markers, CD3 (Leu4) as T-cell marker, and CD10 (J5), CD19 (Leu12) and CD20 (Leu16) as B-cell markers. HLA-DR was detected by OKIal. Anti-Tac and Mik-/31 were used to detect IL-2Ro: and/3, respectively. Fluorescein-conjugated goat anti-mouse lgG F(ab)2 fraction was used as a second reagent. IL-2R/3 was detected with an avidin-biotin detection system; biotinylated goat anti-mouse lgG (Vector, Burlingame, CA) was used as the second reagent and fluorescein isothiocyanate (FITC) conjugated avidin (Tago, Burlingame, CA) as the third reagent. Anti-Tac, Tp40 and MCS2 were kindly provided by Dr T. Uchiyama (Kyoto University, Kyoto, Japan), Dr R. Ueda (Aichi Cancer Center, Nagoya, Japan) and Dr E. Tatsumi (Kobe University, Kobe, Japan). The Leu and OK series were purchased from Becton Dickinson Laboratories (Mountain View, CA) and Ortho Diagnostic Systems (Raritan, NJ), respectively. The other antibodies were from Coulter Immunology (Hialeah, FL). Cell samples were examined by a flow cytometer (Cytron, Ortho). The criteria for surface marker positivity was expression in at least 15% of the leukemic cell population. Induction of IL-2R MNC were cultured in RPMI 1640 medium sup-
plemented with 10% FCS at a concentration of 2 x 106/ml with and without 10U/ml of recombinant human interleukin-1/3 (rhIL-lfl; kindly provided by Otsuka Pharmaceutical Co., Tokyo, Japan) at 37°C in a humidified 5% CO2 incubator. After 48 h the cells were harvested and the expression of IL-2Ro~ and fl was analyzed by flow cytometry as described above. Results Induction of IL-2R
The induction of IL-2Rol and IL-2Rfl in non-T lymphoid leukemia is shown in Tables 1 and 2. IL2R on the contaminated normal T-cells were judged by assessing the expression of the mature T-cell antigen, CD3. In fact, the contaminated T-cells comprised less than 5% of the leukemic cell fraction from each sample. In the presence of rhIL-1/3, IL-2Rcrwas induced on leukemic cells from six of seven cases (2/2 CD19 + C D 1 0 - A L L , 1/2 c o m m o n A L L , 2/2 BCLL and 1/1 leukemic phase of n o n - H o d g k i n ' s lymphoma). The same six and the other CD19 + CD10-
Table 2. Results of liquid culture in IL-2Rcr-inducible cases Fresh cells
After culture IL-1/3 ( - )
Case Pre-B ALL 1. 2. 3. 4. B-CLL 1. 2. LP of lymphoma 1.
IL-1/3 (+)
o:
/3
CD13
o~
/3
CD13
ol
/3
CD13
0 7 0 29
NT 0 0 0
12 2 0 0
37 70 16 40
NT 0 0 NT
54 56 40 NT
NT 54 41 43
NT 0 0 NT
NT 58 40 NT
8 8
0 NT
0 10
68 52
0 0
NT 10
62 52
0 0
NT 10
16
0
0
87
NT
NT
87
NT
NT
Data are given as percentage of positive cells. o6 IL-2Ro~; fl, IL-2Rfl; NT, not tested; LP of lymphoma, leukemic phase of non-Hodgkin's lymphoma.
Induction of IL-2R on non-T lymphoid leukemia
857
Table 3. Immunophenotype of pre-B ALL cells Case
CD10
CD19
CD20
CD3
CD11b
CD13
CD33
CD34
DR
97 50 95 94
1 0 0 86
0 0 0 1
10 8 13 5
12 2 0 l
7 39 6 2
91 NT 93 88
90 94 95 90
27 87
14 1
3 0
NT 0
0 0
0 1
0 0
74 75
IL-2R~ inducible case 1. 0 2. 3 3. 0 4. 95 IL-2R~ uninducible case 5. 7 6. 39
Data are given as percentage of positive cells. NT, not tested; DR, HLA-DR. (A) Fresh cells 100 80 60 40 20
I_A
0
°Jk,
Cont.
IL-2R~t
CDI 3
( B ) C u h e r e d cells w i t h o u t I L - I ~ 100 80' 60
/
4o
I~/J';~, .....
20 0 Cont.
(C
t
t .......
IL-2R~
CDI3
IL-2R~
CD13
Cultered cells with I L - I ~
100 80 60
04°/11 Cont.
Fig. 1. Histogram of flow cytometric analysis of IL-2Rol and CD13 expression in cultured cells with and without IL-lfi. Leukemic cells from a patient with pre-B ALL were cultured for 48 h. (A) Neither IL-2Ra nor CD13 was expressed on fresh cells. (B) CD13 was induced and IL2Ro~ was slightly induced in cultured cells without IL-lfi. (C) Both CD13 and IL-2Ra" were clearly induced in the presence of IL-lfi. A L L case s h o w e d IL-2Rcr induction even without rhIL-lfi. I L - 2 R f i did not a p p e a r on leukemic cells f r o m any of the five cases tested.
Cellular characteristics of IL-2 oMnducible pre-B A L L cells Stem cell antigen C D 3 4 was detected in all three
e x a m i n e d IL-2Rc~-inducible pre-B A L L cases, but in neither of its two uninducible cases (Table 3). T h r e e cases examined could simultaneously induce myeloid associated antigen C D 1 3 with IL-2Ro~(Table 2). T w o IL-2Roluninducible cases e x a m i n e d could not induce CD13. Figure 1 shows the expression of C D 1 3 and IL-2Rcrin the pre-B A L L case (case 2) after culture.
858
K. Nakase et al.
Discussion In the present study, we demonstrated that IL-2Rc~ but not IL-2Rfl was inducible on non-T lymphoid leukemia cells after culture with and without IL-lfl. IL-2Rcrexpression was described in a significant proportion of non-T lymphoid leukemia cases [8, 11] and its induction was also reported in some cases of preB A L L and B-CLL in vitro with phorbol ester or phytohemagglutinin, a potent but non-physiological stimulator [12, 13]. In contrast to previous reports [13, 14], we performed culture experiments using a common physiological activator, IL-1/3 to determine the inducibility of IL-2R. Little is known about the induction of IL-2Rfl expression on lymphoid leukemia cells. Our data indicate that IL-2Rflexpression could not be induced on non-T lymphoid leukemia cells with IL-lfl at least at a detectable level of flow cytometric analysis. On the other hand, IL-lfl was an inducer of IL-2Rol on leukemic cells in most of our cases. Namely, IL-2Ra~ was induced in all cases examined of mature B-cell disorders, B-CLL and leukemic phase of non-Hodgkin's lymphoma and in some cases of pre-B ALL. Interestingly, IL-2Rct inducibility was observed even in the absence of IL-1/3. In T-cell lymphoid leukemia, IL-2Rct expression was rare [15] except in adult T-cell leukemia lymphoma [16] and its induction was demonstrated only in certain cases treated with potent inducers [17, 18]. These facts suggest that IL-2R0t expression might be more regulated in T-cell leukemia than in non-T leukemia. In this study, we found that IL-2Roz-inducible pre-B A L L cells showed common phenotypic characteristics. In comparison with IL-2Rol-uninducible pre-B A L L cells, such cells displayed stem cell antigen CD34 and could induce myeloid-associated antigen CD13. This finding suggests that IL-2Rcr-inducible cells originate from lymphoid myeloid progenitor cells or more immature cells. Thus, in non-T lineage, certain cases of mature B-cell disorders, CLL and leukemic phase of nonHodgkin's lymphoma and pre-B A L L with immature characteristics seem to possess a high potentiality of cell surface IL-2R~r induction. We do not know the biological significance of the induction of IL-2Rcr without IL-2Rfl which is responsible for signal transduction. As for pre-B A L L , however, we have recently shown that CD13 positive cases frequently expressed IL-2Rtr [15]. Therefore, the simultaneous induction of IL-2Ro~ and CD13 on pre-B A L L cells in this study was very interesting. Arita et al. reported the same findings on immature T-cell leukemia cells [18]. In myelocytic leukemia cells, IL-2Ra, was shown to be involved in cell-to-cell interactions [19]. Since CD13 is a cell surface endopeptidase [20], IL-2Ra~might co-operate
with CD13 in cell-to-cell interactions at an early stage of lymphopoiesis. In order to verify this issue, further investigations of biological roles of IL-2Rcr and various cell-to-cell interactions in hematopoiesis should be carried out. Acknowledgements--We thank Drs T. Uchiyama and E. Tatsumi for supplying monoclonal antibodies. We also thank Drs T. Kyo and H. Dohy, Hiroshima Red Cross Hospital, Hiroshima; K. Nasu, Osaka Red Cross Hospital, Osaka; T. Ueda and H. Tsutani, Fukui Medical School, Fukui for referring patient material; and K. Hirose and J. Saitoh for technical assistance. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, and Grantsin-Aid for Cancer Research from the Ministry of Health and Welfare of Japan.
References 1. Morgan D. A., Ruscetti F. W. & Gallo R. C. (1976) Selective in vitro growth of T-lymphocytes from normal human bone marrows. Science 193, 1007. 2. Cantrel D. A. & Smith K. A. (1984) The interleukin 2 T-cell system: a new cell growth model. Science 224, 1312. 3. Takeshita T., Asao H., Ohtani K., Ishii N., Kumaki S., Tanaka N., Munakata H., Nakamura M. & Sugamura K. (1992) Cloning of the y chain of the human IL-2 receptor. Science 257, 379. 4. Faure G. C., Bene M-C. & Bolle-Chanal M-H. (1987) Expression of CD25 (Tac antigen) in lymphoid leukemias and non-Hodgkin lymphomas. Eur. J. Haemat. 38, 26. 5. Erber W. N., Phil D. & Mason D. Y. (1988) Expression of the interleukin-2 receptor (Tac antigen / CD25) in hematologic neoplasms. Am. J, clin. Path. 89, 645. 6. Motoi T., Uchiyama T., Hori T., Itoh K., Uchino H. & Ueda R. (1989) Elevated serum-soluble interleukin2 receptor (Tac antigen) levels in chronic myelogenous leukemia patients with blastic crisis. Blood 74, 1052. 7. Padros M. R., Salamone M. C., Zunszain P. A. & Fainboim L. (1989) Differential expression of CD25 and HC2 antigen subtypes of acute myeloid leukemias. Eur. J. Haemat. 42, 436. 8. Nakase K., Kita K., Otsuji A., Anazawa H., Hoshino K., Sekine T., Shirakawa S., Tanaka I., Nasu K., Tsutani H., Dohy H. & Tsudo M. (1992) Diagnostic and clinical importance of interleukin-2 receptor alpha chain expression on non-T-cell acute leukaemia cells. Br. J. Haemat. 80, 317. 9. Nakase K., Kita K., Anazawa H., Hoshino K., Shirakawa S., Tanaka I. & Tsudo M. (1994) Induction of interleukin-2 receptor alpha chain expression on immature acute myelocytic leukemia cells. Leukemia Res. 18, 269. 10. Kita K., Nasu K., Kamesaki H., Doi S., Tezuka H., Tatsumi E., Fukuhara S., Nishikori M., Uchino H. & Shirakawa S. (1985) Phenotypic analysis of acute lymphoblastic leukemia (ALL) cells which are classified as non-T, non-B and negative for common ALL antigen. Blood 66, 47. 11. Nakase K,, Kita K., Nasu K., Ueda T., Tanaka I,,
Induction of IL-2R on non-T lymphoid leukemia
12.
13.
14.
15.
Shirakawa S. & Tsudo M. (1994) Differential expression of interleukin-2 receptors (a and/3 chain) in mature lymphoid neoplasms. A m . J. Hemat. 46, 179. Touw I., Delwel R., Bolhuis R., Zanen G. & Lowenberg B. (1985) Common and pre-B acute lymphoblastic leukemia cells express interleukin 2 receptors, and interleukin 2 stimulates in vitro colony formation. Blood 66, 556. Touw I., Dorssers L. & Lowenberg B. (1987) The proliferative response of B-cell chronic lymphocytic leukemia to interleukin 2: functional characterization of the interleukin 2 membrane receptors. Blood 69, 1667. Matsuoka M., Hattori T., Kawano F., lshii T., Uchiyama T. & Takatsuki K. (1986) Expression of Tac antigen on human immature B-cell lineage leukemic cells. Leukemia Res. 10, 597. Nakase K., Kita K., Kageyama S., Tsuji K., Miyanishi E., Miwa H., Tanaka lz., Shirakawa S., Tanaka I., Anazawa H., Tsutani H., Kyo T., Dohy H., Kamada N., Doi S., Nasu K. & Uchino H. (1994) Clinical importance of interleukin-2 receptor alpha chain expression in acute leukemia. Cancer Detect. Prey. (in press).
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16. Uchiyama T., Hori T., Tsudo M., Wano Y., Umadome H., Tamori S., Yodoi J., Maeda M., Sawami H. & Uchino H. (1985) Interleukin 2 receptor (Tac antigen) expressed on adult T-cell leukemia cells. J. clin. Invest. 76, 446. 17. Morishima Y., Morishita Y., Adachi K., Tanimoto M., Ohno R. & Saito H. (1987) Phorbol ester induces interleukin-2 receptor on the cell surface of precursor thymocyte leukemia with no rearrangement of T cell receptor fi and 7 genes. Blood 70, 1291. 18. Arita Y., Kita K., Nasu K., Doi S., Fukuhara S., Nishikori M., Miwa H., Tatsumi E., Nosaka T., Hatanaka M. & Uchino H. (1990) Frequent expression of myeloid antigen (CD13) on immature T cells in culture. Acta Hemat. Jpn. 53, 21. 19. Nakase K., Kita K., Otsuji A., Anazawa H., Shirakawa S., Nasu K., Dohy H., Tsutani H. & Tanaka I. (1993) Interleukin 2 receptor alpha chain on acute myelocytic leukemia cells is involved in cell-to-cell interactions. Leukemia Res. 17, 17. 20. Look A. T., Ashmun R. A., Shapiro L. H. & Peiper S. C. (1989) Human myeloid plasma membrane glycoprotein CD13 (gpl50) is identical to aminopeptidase N. J. clin. Invest. 83, 1299.
Leukemia Research Vol. 18, No. 11, pp. 855-859, 1994 Elsevier Science Ltd Printed in Great Britain 0145 2126,/94 $7.00 + 0.00
0145-2126(94)00099-9
INDUCTION OF CELL SURFACE INTERLEUKIN 2 RECEPTOR ALPHA CHAIN EXPRESSION ON NON-T LYMPHOID LEUKEMIA CELLS Kazunori Nakase,* Kenkichi Kita,t Shigeru Shirakawa,t Isao Tanaka,$ and Mitsuru Tsudo§ *The Department of Internal Medicine, Yamada Red Cross Hospital, Misono, Japan; tThe Second Department of Internal Medicine, Mie University School of Medicine, Tsu, Japan; SThe Department of Internal Medicine, Suzuka Kaisei Hospital, Suzuka, Japan; and §The Department of Internal Medicine, Kyoto Katsura Hospital, Kyoto, Japan
(Received 22 April 1994. Accepted 2 July 1994) Abstract--We examined nine cases of adult non-T lymphoid leukemia to investigate the cell surface inducibility of interleukin 2 receptor achain (IL-2Ro~) and #chain (IL-2R#) after in vitro culture with and without recombinant human interleukin-l# (rhlL-l#). Induction of IL-2R~was observed in four of six cases with precursor B-cell acute lymphoblastic leukemia (pre-B ALL) and in all of three cases with B-cell mature lymphoid neoplasm (two chronic lymphocytic leukemia and one leukemic phase of non-Hodgkin's lymphoma). All of the IL-2Ro~-inducible cases could express this spontaneously even without rhlL-l#, while IL-2R# did not appear on leukemic cells from any of the cases tested. IL-2Ro~-inducible pre-B ALL cases displayed stern cell antigen CD34 and induced myeloid-associated antigen CD13 simultaneously. These results suggest that IL-2Ro~but not IL-2R# is easily inducible in certain cases of mature B-cell lymphoid neoplasm and pre-B ALL with immature characteristics.
Key words: Interleukin 2 receptor alpha chain, non-T lymphoid leukemia, CD34, CD13.
Introduction
indicate that IL-2Rcr but not IL-2R/3 is inducible in mature B-cell lymphoid disorders and precursor Bcell acute lymphoblastic leukemia with immature properties.
Interleukin 2 (IL-2), initially referred to as a T-cell growth factor [1], must bind to a specific cell surface IL-2 receptor (IL-2R) to play its biological role [2]. The IL-2R is formed by three different peptides (o~, /3 and 7 chains) [3]. The expression of IL-2R is demonstrated not only in T-cell lymphocytes but also in various non-T hematologic disorders [4-8]. However, little is known about the regulation of IL2R expression in non-T leukemia cells. We have recently described the easily induced expression of IL-2Rc~ chain (IL-2Rc 0 on immature acute myelocytic leukemia cells [9]. In the present study, we investigated the cell surface inducibility of two subunits of IL-2R ( a and /3 chains) on non-T lymphoid leukemia cells and analyzed the cellular characteristics of its inducible cases in relation to immunophenotype. Our results
Materials and Methods
Patients and diagnosis Nine adult patients (over 15 years old) with non-T lymphoid leukemia were referred for this study. The diagnoses were as follows; six precursor B-cell acute lymphoblastic leukemia (pre-B ALL) (four CD19 + CD10-ALL and two common ALL), two B-cell chronic lymphocytic leukemia (B-CLL) and one leukemic phase of non-Hodgkin's lymphoma. hnrnunophenotyping Mononuclear cells (MNC) were obtained from bone marrow or peripheral blood by Ficoll-Hypaque density gradient centrifugation. In some cases, the separated MNC were frozen in liquid nitrogen with 20% heat inactivated fetal calf serum (FCS, GIBCO Lab., Chargrin Falls) and 10% dimethylsulfoxide, and thawed immediately before use. In order to prevent non-specific binding of monoclonal antibody (mAb) with receptor for the IgG Fc portion, the samples were pretreated in Hank's medium supplemented with 5% heat inactivated human AB serum. Cells were stained by indirect immunofluorescence, as previously described [10]. CDllb (OKM1), CD13 (MCS2), CD33
Abbreviations: IL-2Rcr, interleukin 2 receptor alpha chain; IL-2R#, interleukin 2 receptor beta chain; ALL, acute lymphoblastic leukemia; CLL, chronic lymphocytic leukemia; IL-I#, interleukin 1#. Correspondence to: Kazunori Nakase, Department of Internal Medicine, Yamada Red Cross Hospital, Misono 810, Mie 516, Japan (Tel: 596-28-2171; Fax 596-28-2965). 855
856
K. Nakase et al. Table 1. Induction of IL-2R on non-T lymphoid leukemia cells IL-1/3 ( - ) Pre-B CD19 (+) CD10 ( - ) ALL Common ALL Mature B CLL LP of lymphoma Total
IL-1/3 (+)
IL-2Ro:
1L-2R/3
IL-2Ro:
IL-2R/3
3/4 1/2
0/3 0/1
2/2 1/2
0/2 0/1
2/2 1/1 7/9
0/2 NT 0/6
2/2 1/1 6/7
0/2 NT 0/5
Number of IL-2R inducible cases / number of cases tested. LP of lymphoma, leukemic phase of non-Hodgkin's lymphoma; NT, not tested.
(My9) and CD34 (My10) were tested as myeloid markers, CD3 (Leu4) as T-cell marker, and CD10 (J5), CD19 (Leu12) and CD20 (Leu16) as B-cell markers. HLA-DR was detected by OKIal. Anti-Tac and Mik-/31 were used to detect IL-2Ro: and/3, respectively. Fluorescein-conjugated goat anti-mouse lgG F(ab)2 fraction was used as a second reagent. IL-2R/3 was detected with an avidin-biotin detection system; biotinylated goat anti-mouse lgG (Vector, Burlingame, CA) was used as the second reagent and fluorescein isothiocyanate (FITC) conjugated avidin (Tago, Burlingame, CA) as the third reagent. Anti-Tac, Tp40 and MCS2 were kindly provided by Dr T. Uchiyama (Kyoto University, Kyoto, Japan), Dr R. Ueda (Aichi Cancer Center, Nagoya, Japan) and Dr E. Tatsumi (Kobe University, Kobe, Japan). The Leu and OK series were purchased from Becton Dickinson Laboratories (Mountain View, CA) and Ortho Diagnostic Systems (Raritan, NJ), respectively. The other antibodies were from Coulter Immunology (Hialeah, FL). Cell samples were examined by a flow cytometer (Cytron, Ortho). The criteria for surface marker positivity was expression in at least 15% of the leukemic cell population. Induction of IL-2R MNC were cultured in RPMI 1640 medium sup-
plemented with 10% FCS at a concentration of 2 x 106/ml with and without 10U/ml of recombinant human interleukin-1/3 (rhIL-lfl; kindly provided by Otsuka Pharmaceutical Co., Tokyo, Japan) at 37°C in a humidified 5% CO2 incubator. After 48 h the cells were harvested and the expression of IL-2Ro~ and fl was analyzed by flow cytometry as described above. Results Induction of IL-2R
The induction of IL-2Rol and IL-2Rfl in non-T lymphoid leukemia is shown in Tables 1 and 2. IL2R on the contaminated normal T-cells were judged by assessing the expression of the mature T-cell antigen, CD3. In fact, the contaminated T-cells comprised less than 5% of the leukemic cell fraction from each sample. In the presence of rhIL-1/3, IL-2Rcrwas induced on leukemic cells from six of seven cases (2/2 CD19 + C D 1 0 - A L L , 1/2 c o m m o n A L L , 2/2 BCLL and 1/1 leukemic phase of n o n - H o d g k i n ' s lymphoma). The same six and the other CD19 + CD10-
Table 2. Results of liquid culture in IL-2Rcr-inducible cases Fresh cells
After culture IL-1/3 ( - )
Case Pre-B ALL 1. 2. 3. 4. B-CLL 1. 2. LP of lymphoma 1.
IL-1/3 (+)
o:
/3
CD13
o~
/3
CD13
ol
/3
CD13
0 7 0 29
NT 0 0 0
12 2 0 0
37 70 16 40
NT 0 0 NT
54 56 40 NT
NT 54 41 43
NT 0 0 NT
NT 58 40 NT
8 8
0 NT
0 10
68 52
0 0
NT 10
62 52
0 0
NT 10
16
0
0
87
NT
NT
87
NT
NT
Data are given as percentage of positive cells. o6 IL-2Ro~; fl, IL-2Rfl; NT, not tested; LP of lymphoma, leukemic phase of non-Hodgkin's lymphoma.
Induction of IL-2R on non-T lymphoid leukemia
857
Table 3. Immunophenotype of pre-B ALL cells Case
CD10
CD19
CD20
CD3
CD11b
CD13
CD33
CD34
DR
97 50 95 94
1 0 0 86
0 0 0 1
10 8 13 5
12 2 0 l
7 39 6 2
91 NT 93 88
90 94 95 90
27 87
14 1
3 0
NT 0
0 0
0 1
0 0
74 75
IL-2R~ inducible case 1. 0 2. 3 3. 0 4. 95 IL-2R~ uninducible case 5. 7 6. 39
Data are given as percentage of positive cells. NT, not tested; DR, HLA-DR. (A) Fresh cells 100 80 60 40 20
I_A
0
°Jk,
Cont.
IL-2R~t
CDI 3
( B ) C u h e r e d cells w i t h o u t I L - I ~ 100 80' 60
/
4o
I~/J';~, .....
20 0 Cont.
(C
t
t .......
IL-2R~
CDI3
IL-2R~
CD13
Cultered cells with I L - I ~
100 80 60
04°/11 Cont.
Fig. 1. Histogram of flow cytometric analysis of IL-2Rol and CD13 expression in cultured cells with and without IL-lfi. Leukemic cells from a patient with pre-B ALL were cultured for 48 h. (A) Neither IL-2Ra nor CD13 was expressed on fresh cells. (B) CD13 was induced and IL2Ro~ was slightly induced in cultured cells without IL-lfi. (C) Both CD13 and IL-2Ra" were clearly induced in the presence of IL-lfi. A L L case s h o w e d IL-2Rcr induction even without rhIL-lfi. I L - 2 R f i did not a p p e a r on leukemic cells f r o m any of the five cases tested.
Cellular characteristics of IL-2 oMnducible pre-B A L L cells Stem cell antigen C D 3 4 was detected in all three
e x a m i n e d IL-2Rc~-inducible pre-B A L L cases, but in neither of its two uninducible cases (Table 3). T h r e e cases examined could simultaneously induce myeloid associated antigen C D 1 3 with IL-2Ro~(Table 2). T w o IL-2Roluninducible cases e x a m i n e d could not induce CD13. Figure 1 shows the expression of C D 1 3 and IL-2Rcrin the pre-B A L L case (case 2) after culture.
858
K. Nakase et al.
Discussion In the present study, we demonstrated that IL-2Rc~ but not IL-2Rfl was inducible on non-T lymphoid leukemia cells after culture with and without IL-lfl. IL-2Rcrexpression was described in a significant proportion of non-T lymphoid leukemia cases [8, 11] and its induction was also reported in some cases of preB A L L and B-CLL in vitro with phorbol ester or phytohemagglutinin, a potent but non-physiological stimulator [12, 13]. In contrast to previous reports [13, 14], we performed culture experiments using a common physiological activator, IL-1/3 to determine the inducibility of IL-2R. Little is known about the induction of IL-2Rfl expression on lymphoid leukemia cells. Our data indicate that IL-2Rflexpression could not be induced on non-T lymphoid leukemia cells with IL-lfl at least at a detectable level of flow cytometric analysis. On the other hand, IL-lfl was an inducer of IL-2Rol on leukemic cells in most of our cases. Namely, IL-2Ra~ was induced in all cases examined of mature B-cell disorders, B-CLL and leukemic phase of non-Hodgkin's lymphoma and in some cases of pre-B ALL. Interestingly, IL-2Rct inducibility was observed even in the absence of IL-1/3. In T-cell lymphoid leukemia, IL-2Rct expression was rare [15] except in adult T-cell leukemia lymphoma [16] and its induction was demonstrated only in certain cases treated with potent inducers [17, 18]. These facts suggest that IL-2R0t expression might be more regulated in T-cell leukemia than in non-T leukemia. In this study, we found that IL-2Roz-inducible pre-B A L L cells showed common phenotypic characteristics. In comparison with IL-2Rol-uninducible pre-B A L L cells, such cells displayed stem cell antigen CD34 and could induce myeloid-associated antigen CD13. This finding suggests that IL-2Rcr-inducible cells originate from lymphoid myeloid progenitor cells or more immature cells. Thus, in non-T lineage, certain cases of mature B-cell disorders, CLL and leukemic phase of nonHodgkin's lymphoma and pre-B A L L with immature characteristics seem to possess a high potentiality of cell surface IL-2R~r induction. We do not know the biological significance of the induction of IL-2Rcr without IL-2Rfl which is responsible for signal transduction. As for pre-B A L L , however, we have recently shown that CD13 positive cases frequently expressed IL-2Rtr [15]. Therefore, the simultaneous induction of IL-2Ro~ and CD13 on pre-B A L L cells in this study was very interesting. Arita et al. reported the same findings on immature T-cell leukemia cells [18]. In myelocytic leukemia cells, IL-2Ra, was shown to be involved in cell-to-cell interactions [19]. Since CD13 is a cell surface endopeptidase [20], IL-2Ra~might co-operate
with CD13 in cell-to-cell interactions at an early stage of lymphopoiesis. In order to verify this issue, further investigations of biological roles of IL-2Rcr and various cell-to-cell interactions in hematopoiesis should be carried out. Acknowledgements--We thank Drs T. Uchiyama and E. Tatsumi for supplying monoclonal antibodies. We also thank Drs T. Kyo and H. Dohy, Hiroshima Red Cross Hospital, Hiroshima; K. Nasu, Osaka Red Cross Hospital, Osaka; T. Ueda and H. Tsutani, Fukui Medical School, Fukui for referring patient material; and K. Hirose and J. Saitoh for technical assistance. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, and Grantsin-Aid for Cancer Research from the Ministry of Health and Welfare of Japan.
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