In VitroCell.Dev.Biol.28A:657-662,September-October1992 © 1992TissueCultureAssociation 0883-8364/92 $01.50+0.O0
COLONYSTIMULATING FACTOR, TUMOR NECROSIS FACTOR-a, AND INTERFERON-7 INDUCIBLE
EXPRESSION
IN TWO
HUMAN
OF
GRANULOCYTE-MACROPHAGE
CYTOTOXIC
LEUKEMIC
T-CELL
LINES
ALESSANDRA CESANO AND DANIELA SANTOLI1
The Wistar Institute of Anatomy and Biology., 3601 Spruce Street, Philadelphia, Pennsylvania 19104 (Received 7 April 1992; accepted 1 May 1992)
SUMMARY We investigated the ability of the TALL-103/2 and TALL-104 leukemic cell lines to produce lymphokines in response to activation signals, such as tumor cells and anti-CD3 (OKT3) or -CD2 (B67.1) monoclonal antibodies (mAb) or both. Both cell lines were found to produce high levels of interferon (IFN)-% tumor necrosis factor (TNF)-(x, and granulocytemacrophage colony-stimulatingfactor (GM-CSF). The latter lymphokine is induced by lysable tumor cells and by immobilized OKT3 and B67.1 mAb only in the presence of interleukin (IL-2). 1FN-'y and TNF-o~ are induced upon CD3 but not CD2 stimulation, both in the presence and absence of IL-2. Interestingly, the B67.1 mAb amplifies the OKT3-induced responses by 2- to lO-fold, bringing the IFN-~ and TNF-o~ levels of production up to 200 U/ml. Thus, simultaneous triggering of the CD2 and CD3 signaling pathways results in a very efficient lymphokine release. Of all the tumor cell lines tested as inducers, only K562 cells are able to stimulate the production of IFN-'y and TNF-o~ in TALL-103/2 and TALL-104 cells, especially upon culture in IL-2. Lymphokine mRNA expression after stimulation with mAb or K562 cells peaks at 2 h in both cell lines. No messages are detectable in TALL-103/2 ceils at 8 h, whereas in TALL-104 cells, IFN-"y and GM-CSF transcripts are still present at 8 and 20 h, respectively. The inducible and highly regntatabte expression of lymphokine release by these cell lines provides a unique model for studying mechanisms of lymphokine induction by different biological agents.
Key words: cytotoxic T cell lines; lymphokine induction; tumor targets; interferon-v; tumor necrosis factor-a; GM-CSF. phokine release, to our knowledge the cytotoxic cells did not represent clonal populations. The present investigation demonstrates that homogeneous CD8 + human leukemic T-cell lines, endowed with the major histocompatibility complex nonrestricted cytotoxic function [see accompanying manuscript (5)], rapidly and efficiently release high levels of at least three lymphokines upon specific triggering of the CD2 and CD3 signaling pathways or co-culture with lysable tumor cells.
INTRODUCTION Several studies have demonstrated the ability of various types of activated effector cells to produce lymphokines which arc important in inflammation and immune responses. Purified large granular lymphocytes (LGL) have been shown to produce interleukin (IL-2), interferon (IFN)-'y, and colony-stimulating factor (CSF) (13). LGL are also induced by natural killer (NK)-sensitive tumor cell lines to produce tumor necrosis factor (TNF)-o~, although this factor does not seem to be responsible for cytotoxicity against classical NK target cells such as K562 or Moh-4 (21). The ability of human peripheral blood lymphocytes, depleted of monocytes, to produce high levels of TNF when stimulated with phorbo[ diester and calcium ionophore, or with mitogen, was also shown (9). Like IL-2 and IFN-'y, TNF-o~ has multiple immunoregulatory activities, which include activation of NK cells and macrophages (18,22), and induction of growth and function of B and T lymphocytes in vitro (14,24). Also, IFN-'y and TNF-a synergize with IL-2 to augment cytotoxic activity of human cytotoxic T lymphocytes and lymphokine-actirated killer (LAK) cells (12,16,19,26). Although in most reports the effector cell populations were highly purified and carefully depleted of contaminating cells, which could have been directly or indirectly responsible for the observed lym-
MATERIALSAND METHODS
Cell likes. The IL-2-dependent TALL-103/2 (CD8+CD3+TCR'y6+) and TALL-104 (CD8+CD3+TCRo~+) cell lines, the leukemic Raji, Daudi, K562, U937, and HL60 cells, and the murine Fc receptor-positive (FcR+) P815 cells have been described in the companion manuscript (5). The human megakaryoblastic leukemia cell line, MO7E (2), was obtained from Dr. Luigi Pegoraro (University of Torino, Italy) and maintained at 37 ° C in Iscove's modified Dulbecco's medium (GIBCO, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum. MOTE cells are strictly dependent on biweekly additions of granulocyte-macrophage (GM)CSF (0.5 ng/ml) for growth and viability. Growthfactors and antibodies. Pure recombinant human (rh)IL-2 (specific activity 7 X 106 U/mg protein) was purchased from Amgen (Thousand Oaks, CA). Homogeneous GM-CSF (Chinese hamster ovary cell-derived, specific activity 1 × 107 U/mg) was a gift from Dr. Steven Clark (The Genetics Institute, Cambridge, MA). Ascites OKT3 (anti-CD3) (Ortho Pharmaceuticals, Raritan, NJ) and B67.1 (ami-CD2) (a gift from Dr. Bice Perussia, Jefferson Medical School, Philadelphia, PA) were used at the dilution of 1:100.
1 To whom correspondence should be addressed. 657
658
CESANO AND SANTOLI
Sheep anti-human GM-CSF was supplied by Dr. S. Clark and proven to be effective in neutralizing specific CSF activity in the chronic myeloid leukemia blast assay. Rabbit anti-human IL-3 and IL-9 and goat anti-human IL-6 were also gifts from the Genetics Institute. Rabbit anti-human IL-4 was purchased from Genzyme (Boston, MA). Goat anti-human IL-2 and monoclonal antibodies (mAb) anti-IFN-'y and -TNF-a were kindly provided by Dr. Giorgio Trinchieri (The Wistar Institute, Philadelphia, PA): All sera were used in proliferation assays at 1 and 5 X 10 -z. Lymphokine induction. TALL-103/2 and TALL-104 cells (1 × 106/ ml) were incubated at 37 ° C for 16 to 20 h in the presence and absence of rhlL-2 (10 U/ml) and other inducers, such as leukemic cell lines (0.2 X 106/ml) or purified mAb (10-2). Cell-free conditioned medium (CM) was harvested, filtered, and tested for the presence of IFN-'y, GM-CSF, and TNF-a as outlined below. When OKT3 (anti-CD3) and B67.1 (anti-CD2) mAb were used as inducers, they were immobilized on 60-mm petri dishes by diluting them at 10 -2 in 5 ml NaCOa/NaHCO 2 buffer; after overnight incubation at 0 ° C, the dishes were washed with medium supplemented with 5% fetal bovine serum and 10 mM HEPES buffer. TALL-I03/2 and TALL-104 cells were then plated (2 × 107/dish) and incubated at 37 ° C in the presence of 10 U/ml IL-2. In some experiments, the effector cells were first coated with mAb, then washed and further incubated with the FcR+ P815 cells (0.2 × 106/ml). Interferon and TNF assays. TNF-a and IFN-~' production was tested by radioimmunoassay as described (15), using mAbs supplied by Dr. G. Trinehieri. Specifically, mAb B154.7 and B154.9 were used as labeled and unlabeled antibody, respectively, to detect the presence of TNF-c~in the CM preparations. TNF-c¢ concentrations were measured based on a standard curve which was derived in each experiment using a recombinant TNF standard. The sensitivity of the assay was at least 0.1 U/mE Production of IFN-'y in radioimmunoassay was detected using mAb B133.1 and B133.5 as first and second antibodies, respectively. The sensitivity of the assay was 1 U/ml of IFN-'y, and no cross-reactivity was observed with TNF, lymphotoxin, nor other species of IFN. Granulocyte macrophage-CSFassays. The ability of TALL-103/2- and TALL-104-CM to stimulate short-term prohferation of the GM-CSF-dependent MO7E cells was investigated. For these experiments, MO7E cells were harvested at the end of their 4-day culture cycle, when most or all of the rhGM-CSF had been consumed, washed, and seeded at 2 × 104/well in 96-well microtiter plates (Falcon, Becton Dickinson, Oxnard, CA) in the presence of 50% CM or the indicated concentrations.of rhGM-CSF. All test samples were or were not adsorbed for 30 rain at room temperature with neutralizing antisera (1 and 5 × 10 -2) specific to human growth factors before being added to the indicator ceils. Control cells were incubated in medium alone. After 3 to 4 days, the cells were pulsed with 1 #Ci [aH]thymidine (2 Ci/mmul) for 6 h, and isotope incorporation was measured as described (17,23). Data are presented as mean counts per minute for triplicate wells. The presence of GM-CSF' in the CM was also quantitated using the Quantikine human GM-CSF immunoassay kit, employing an immunometric "sandwich" enzyme-hnked immunoabsorbent assay (R and D Systems, Minneapohs, MN). The sensitivity of the assay was at least 7 pg/ml. Northern blot analysis. Total cellular RNA from unstimulated and mAb-stimulated TALL-t03/2 and TALL-104 cells was extracted at the indicated times using the RNAzol procedure (8). Northern blot hybridization using cDNA probes for GM-CSF, IFN-'y, and TNF-ot (kindly provided by Dr. G. Trinchieri) was performed as described in the accompanying manuscript. RESULTS
Interferon-3, and TNF-a production by TALL-103/2 and TALL104 cells. Kinetic studies indicated that lymphokine release by TALL-103/2 and TALL-104 cells is not detectable before 8 h of stimulation with several inducers, and that maximum levels are reached after 24 h (data not shown). Table 1 shows the results of a representative experiment in which the 24-h CM of the two leukemic cell lines were analyzed for the presence of constitutive or induced TNF-o~ and IFN-% The" values shown in the table have already been corrected for specificity by subtracting spontaneous lymphokine production by the stimuli.alone (human tumor cells and
murine P815 cells produce < 1 U / m l of each lymphokine, except for K562 cells which constitutively produce up to 2.8 U / m l of TNF-c~). As measured by radioimmunoassay, unstimulated TALL1 0 3 / 2 and TALL-104 cells produce little, if any, IFN-3' and TNFc~ both in the presence and absence of IL-2. Of ail the tumor ceil fines tested (which also represent the cells used as targets for cytotoxic assays in the companion study), K562 is the only one able to induce high levels of both lymphokines in TALL-103/2 cells even in the absence of IL-2. In the case of TALL-104 cells, both K562 and U937 cell lines induce production of IFN-3" (only in the presence of IL-2) and TNF-c~ (both with or without IL-2) at levels significantly lower than those seen in TALL-103/2 cells. Immobilized OKT3 mAb triggers the production of IFN-3" and TNF-a in both celt lines, either in the presence or in the absence of IL-2. This mAb represents a particularly good inducer of IFN-3" in TALL-103/2 cells (up to 146 U/ml). Although by itself the anti-CD2 mAb B67.1 is unable to induce lymphokine release, it does amplify the responses of TALL-103/2 cells to the anti-CD3 mAb, and synergizes with OKT3 in the induction of both lymphokines by TALL-104 cells (up to 200 U/ml of IFN-3" and TNF-a are found in the CM of TALL-104 cells triggered by the combined mAb). Thus, the magnitude of TNF-a and IFN-3" production by these leukemic T cells is remarkably higher upon simultaneous triggering of CD2 and CD3 signaling pathways. Although Table 1 shows only the results obtained when the mAb were immobilized on plates, similar values of IFN-3' and TNF-c~ were induced by mAb crosslinked with FcR + P815 cells (data not shown). In several experiments, immobilized mAb specific for either CD8 (OKT8) or CD56 (anti'-Leu-19) did not stimulate IFN or TNF production in TALL1 0 3 / 2 and TALL-104 cells (not shown). Moreover, when used in a soluble form, none of the mAb tested (including OKT3 and B67.1) was able to induce lymphokine release (not shown). Induction of GM-CSF release. Constitutive and induced GMCSF production by TALL-103/2 and TALL-104 cells was analyzed using a proliferation assay against the highly sensitive MO7E cells as indicators. Figure 1 shows one of three experiments performed, all with similar results, in which the background counts per minute of MO7E cells incubated in the absence of growth factor was 58. Figure 1 A shows the typical dose-dependent proliferative response induced by rhGM-CSF in these cells, and illustrates that even the response induced by the highest concentration of this growth factor (12.8 ng/ml) is totally blocked by pre-incubation with an anti-GM-CSF antibody. TALL-103/2 CM and TALL-104 CM, raised in the absence of IL-2 or any other stimulus, display no detectable stimulatory activity on MO7E cells (B). Of all the tumor target cells tested in the absence of IL-2, K562 was the only one inducing such activity both in TALL-103/2 cells [stimulation index (SI)] (28) and, at much higher levels, in TALL-104 cells (SI 255) (A,B). This activity was totally neutralized by the anti-GM-CSF antibody, but not by antibodies specific for IL-2, IL-3, IL-6, IL-9, TNF-a, or IFN-3" (not shown). TALL-103/2 CM and TALL-104 CM generated in the presence of IL-2 and K562 cells contained higher activity, inducing SI values of 363 and 983, respectively. CM from TALL-104 cells induced by U937 (SI 717), Raji (SI 403), Daudi (SI 252), and HL60 (SI 88) cells also stimulated MO7E cell proliferation (Fig. 1 B). The stimulatory activity of all the CM raised in the presence of IL-2 was completely blocked by the combination of anti-GM-CSF and anti-IL-2 sera (not shown). In the presence of IL-2, the anti-CD2 and, especially, anti-CD3
659
LYMPHOKINE INDUCTION IN T-ALL CELLS TABLE 1
PRODUCTION OF IFN-'y AND TNF-a BY TALL-103/2 AND TALL-104 CELLS UPON STIMULATIONWITH mAb OR TUMOR CELLS TALL-103/2
TALL-104
No IL-2 Stimulus~
A. Tumor cells None K562 U937 Raji Daudi HL60 B. ImmobilizedmAb No mAb OKT3 B67.1 OKT3/B67.1
IL-2
No IL-2
IL~2
IFN-7
TNF-a
IFN-3~
TNF-a
IFN-'y
TNF-a
IFN-3~
TNF-a
COLONYSTIMULATING FACTOR, TUMOR NECROSIS FACTOR-a, AND INTERFERON-7 INDUCIBLE
EXPRESSION
IN TWO
HUMAN
OF
GRANULOCYTE-MACROPHAGE
CYTOTOXIC
LEUKEMIC
T-CELL
LINES
ALESSANDRA CESANO AND DANIELA SANTOLI1
The Wistar Institute of Anatomy and Biology., 3601 Spruce Street, Philadelphia, Pennsylvania 19104 (Received 7 April 1992; accepted 1 May 1992)
SUMMARY We investigated the ability of the TALL-103/2 and TALL-104 leukemic cell lines to produce lymphokines in response to activation signals, such as tumor cells and anti-CD3 (OKT3) or -CD2 (B67.1) monoclonal antibodies (mAb) or both. Both cell lines were found to produce high levels of interferon (IFN)-% tumor necrosis factor (TNF)-(x, and granulocytemacrophage colony-stimulatingfactor (GM-CSF). The latter lymphokine is induced by lysable tumor cells and by immobilized OKT3 and B67.1 mAb only in the presence of interleukin (IL-2). 1FN-'y and TNF-o~ are induced upon CD3 but not CD2 stimulation, both in the presence and absence of IL-2. Interestingly, the B67.1 mAb amplifies the OKT3-induced responses by 2- to lO-fold, bringing the IFN-~ and TNF-o~ levels of production up to 200 U/ml. Thus, simultaneous triggering of the CD2 and CD3 signaling pathways results in a very efficient lymphokine release. Of all the tumor cell lines tested as inducers, only K562 cells are able to stimulate the production of IFN-'y and TNF-o~ in TALL-103/2 and TALL-104 cells, especially upon culture in IL-2. Lymphokine mRNA expression after stimulation with mAb or K562 cells peaks at 2 h in both cell lines. No messages are detectable in TALL-103/2 ceils at 8 h, whereas in TALL-104 cells, IFN-"y and GM-CSF transcripts are still present at 8 and 20 h, respectively. The inducible and highly regntatabte expression of lymphokine release by these cell lines provides a unique model for studying mechanisms of lymphokine induction by different biological agents.
Key words: cytotoxic T cell lines; lymphokine induction; tumor targets; interferon-v; tumor necrosis factor-a; GM-CSF. phokine release, to our knowledge the cytotoxic cells did not represent clonal populations. The present investigation demonstrates that homogeneous CD8 + human leukemic T-cell lines, endowed with the major histocompatibility complex nonrestricted cytotoxic function [see accompanying manuscript (5)], rapidly and efficiently release high levels of at least three lymphokines upon specific triggering of the CD2 and CD3 signaling pathways or co-culture with lysable tumor cells.
INTRODUCTION Several studies have demonstrated the ability of various types of activated effector cells to produce lymphokines which arc important in inflammation and immune responses. Purified large granular lymphocytes (LGL) have been shown to produce interleukin (IL-2), interferon (IFN)-'y, and colony-stimulating factor (CSF) (13). LGL are also induced by natural killer (NK)-sensitive tumor cell lines to produce tumor necrosis factor (TNF)-o~, although this factor does not seem to be responsible for cytotoxicity against classical NK target cells such as K562 or Moh-4 (21). The ability of human peripheral blood lymphocytes, depleted of monocytes, to produce high levels of TNF when stimulated with phorbo[ diester and calcium ionophore, or with mitogen, was also shown (9). Like IL-2 and IFN-'y, TNF-o~ has multiple immunoregulatory activities, which include activation of NK cells and macrophages (18,22), and induction of growth and function of B and T lymphocytes in vitro (14,24). Also, IFN-'y and TNF-a synergize with IL-2 to augment cytotoxic activity of human cytotoxic T lymphocytes and lymphokine-actirated killer (LAK) cells (12,16,19,26). Although in most reports the effector cell populations were highly purified and carefully depleted of contaminating cells, which could have been directly or indirectly responsible for the observed lym-
MATERIALSAND METHODS
Cell likes. The IL-2-dependent TALL-103/2 (CD8+CD3+TCR'y6+) and TALL-104 (CD8+CD3+TCRo~+) cell lines, the leukemic Raji, Daudi, K562, U937, and HL60 cells, and the murine Fc receptor-positive (FcR+) P815 cells have been described in the companion manuscript (5). The human megakaryoblastic leukemia cell line, MO7E (2), was obtained from Dr. Luigi Pegoraro (University of Torino, Italy) and maintained at 37 ° C in Iscove's modified Dulbecco's medium (GIBCO, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum. MOTE cells are strictly dependent on biweekly additions of granulocyte-macrophage (GM)CSF (0.5 ng/ml) for growth and viability. Growthfactors and antibodies. Pure recombinant human (rh)IL-2 (specific activity 7 X 106 U/mg protein) was purchased from Amgen (Thousand Oaks, CA). Homogeneous GM-CSF (Chinese hamster ovary cell-derived, specific activity 1 × 107 U/mg) was a gift from Dr. Steven Clark (The Genetics Institute, Cambridge, MA). Ascites OKT3 (anti-CD3) (Ortho Pharmaceuticals, Raritan, NJ) and B67.1 (ami-CD2) (a gift from Dr. Bice Perussia, Jefferson Medical School, Philadelphia, PA) were used at the dilution of 1:100.
1 To whom correspondence should be addressed. 657
658
CESANO AND SANTOLI
Sheep anti-human GM-CSF was supplied by Dr. S. Clark and proven to be effective in neutralizing specific CSF activity in the chronic myeloid leukemia blast assay. Rabbit anti-human IL-3 and IL-9 and goat anti-human IL-6 were also gifts from the Genetics Institute. Rabbit anti-human IL-4 was purchased from Genzyme (Boston, MA). Goat anti-human IL-2 and monoclonal antibodies (mAb) anti-IFN-'y and -TNF-a were kindly provided by Dr. Giorgio Trinchieri (The Wistar Institute, Philadelphia, PA): All sera were used in proliferation assays at 1 and 5 X 10 -z. Lymphokine induction. TALL-103/2 and TALL-104 cells (1 × 106/ ml) were incubated at 37 ° C for 16 to 20 h in the presence and absence of rhlL-2 (10 U/ml) and other inducers, such as leukemic cell lines (0.2 X 106/ml) or purified mAb (10-2). Cell-free conditioned medium (CM) was harvested, filtered, and tested for the presence of IFN-'y, GM-CSF, and TNF-a as outlined below. When OKT3 (anti-CD3) and B67.1 (anti-CD2) mAb were used as inducers, they were immobilized on 60-mm petri dishes by diluting them at 10 -2 in 5 ml NaCOa/NaHCO 2 buffer; after overnight incubation at 0 ° C, the dishes were washed with medium supplemented with 5% fetal bovine serum and 10 mM HEPES buffer. TALL-I03/2 and TALL-104 cells were then plated (2 × 107/dish) and incubated at 37 ° C in the presence of 10 U/ml IL-2. In some experiments, the effector cells were first coated with mAb, then washed and further incubated with the FcR+ P815 cells (0.2 × 106/ml). Interferon and TNF assays. TNF-a and IFN-~' production was tested by radioimmunoassay as described (15), using mAbs supplied by Dr. G. Trinehieri. Specifically, mAb B154.7 and B154.9 were used as labeled and unlabeled antibody, respectively, to detect the presence of TNF-c~in the CM preparations. TNF-c¢ concentrations were measured based on a standard curve which was derived in each experiment using a recombinant TNF standard. The sensitivity of the assay was at least 0.1 U/mE Production of IFN-'y in radioimmunoassay was detected using mAb B133.1 and B133.5 as first and second antibodies, respectively. The sensitivity of the assay was 1 U/ml of IFN-'y, and no cross-reactivity was observed with TNF, lymphotoxin, nor other species of IFN. Granulocyte macrophage-CSFassays. The ability of TALL-103/2- and TALL-104-CM to stimulate short-term prohferation of the GM-CSF-dependent MO7E cells was investigated. For these experiments, MO7E cells were harvested at the end of their 4-day culture cycle, when most or all of the rhGM-CSF had been consumed, washed, and seeded at 2 × 104/well in 96-well microtiter plates (Falcon, Becton Dickinson, Oxnard, CA) in the presence of 50% CM or the indicated concentrations.of rhGM-CSF. All test samples were or were not adsorbed for 30 rain at room temperature with neutralizing antisera (1 and 5 × 10 -2) specific to human growth factors before being added to the indicator ceils. Control cells were incubated in medium alone. After 3 to 4 days, the cells were pulsed with 1 #Ci [aH]thymidine (2 Ci/mmul) for 6 h, and isotope incorporation was measured as described (17,23). Data are presented as mean counts per minute for triplicate wells. The presence of GM-CSF' in the CM was also quantitated using the Quantikine human GM-CSF immunoassay kit, employing an immunometric "sandwich" enzyme-hnked immunoabsorbent assay (R and D Systems, Minneapohs, MN). The sensitivity of the assay was at least 7 pg/ml. Northern blot analysis. Total cellular RNA from unstimulated and mAb-stimulated TALL-t03/2 and TALL-104 cells was extracted at the indicated times using the RNAzol procedure (8). Northern blot hybridization using cDNA probes for GM-CSF, IFN-'y, and TNF-ot (kindly provided by Dr. G. Trinchieri) was performed as described in the accompanying manuscript. RESULTS
Interferon-3, and TNF-a production by TALL-103/2 and TALL104 cells. Kinetic studies indicated that lymphokine release by TALL-103/2 and TALL-104 cells is not detectable before 8 h of stimulation with several inducers, and that maximum levels are reached after 24 h (data not shown). Table 1 shows the results of a representative experiment in which the 24-h CM of the two leukemic cell lines were analyzed for the presence of constitutive or induced TNF-o~ and IFN-% The" values shown in the table have already been corrected for specificity by subtracting spontaneous lymphokine production by the stimuli.alone (human tumor cells and
murine P815 cells produce < 1 U / m l of each lymphokine, except for K562 cells which constitutively produce up to 2.8 U / m l of TNF-c~). As measured by radioimmunoassay, unstimulated TALL1 0 3 / 2 and TALL-104 cells produce little, if any, IFN-3' and TNFc~ both in the presence and absence of IL-2. Of ail the tumor ceil fines tested (which also represent the cells used as targets for cytotoxic assays in the companion study), K562 is the only one able to induce high levels of both lymphokines in TALL-103/2 cells even in the absence of IL-2. In the case of TALL-104 cells, both K562 and U937 cell lines induce production of IFN-3" (only in the presence of IL-2) and TNF-c~ (both with or without IL-2) at levels significantly lower than those seen in TALL-103/2 cells. Immobilized OKT3 mAb triggers the production of IFN-3" and TNF-a in both celt lines, either in the presence or in the absence of IL-2. This mAb represents a particularly good inducer of IFN-3" in TALL-103/2 cells (up to 146 U/ml). Although by itself the anti-CD2 mAb B67.1 is unable to induce lymphokine release, it does amplify the responses of TALL-103/2 cells to the anti-CD3 mAb, and synergizes with OKT3 in the induction of both lymphokines by TALL-104 cells (up to 200 U/ml of IFN-3" and TNF-a are found in the CM of TALL-104 cells triggered by the combined mAb). Thus, the magnitude of TNF-a and IFN-3" production by these leukemic T cells is remarkably higher upon simultaneous triggering of CD2 and CD3 signaling pathways. Although Table 1 shows only the results obtained when the mAb were immobilized on plates, similar values of IFN-3' and TNF-c~ were induced by mAb crosslinked with FcR + P815 cells (data not shown). In several experiments, immobilized mAb specific for either CD8 (OKT8) or CD56 (anti'-Leu-19) did not stimulate IFN or TNF production in TALL1 0 3 / 2 and TALL-104 cells (not shown). Moreover, when used in a soluble form, none of the mAb tested (including OKT3 and B67.1) was able to induce lymphokine release (not shown). Induction of GM-CSF release. Constitutive and induced GMCSF production by TALL-103/2 and TALL-104 cells was analyzed using a proliferation assay against the highly sensitive MO7E cells as indicators. Figure 1 shows one of three experiments performed, all with similar results, in which the background counts per minute of MO7E cells incubated in the absence of growth factor was 58. Figure 1 A shows the typical dose-dependent proliferative response induced by rhGM-CSF in these cells, and illustrates that even the response induced by the highest concentration of this growth factor (12.8 ng/ml) is totally blocked by pre-incubation with an anti-GM-CSF antibody. TALL-103/2 CM and TALL-104 CM, raised in the absence of IL-2 or any other stimulus, display no detectable stimulatory activity on MO7E cells (B). Of all the tumor target cells tested in the absence of IL-2, K562 was the only one inducing such activity both in TALL-103/2 cells [stimulation index (SI)] (28) and, at much higher levels, in TALL-104 cells (SI 255) (A,B). This activity was totally neutralized by the anti-GM-CSF antibody, but not by antibodies specific for IL-2, IL-3, IL-6, IL-9, TNF-a, or IFN-3" (not shown). TALL-103/2 CM and TALL-104 CM generated in the presence of IL-2 and K562 cells contained higher activity, inducing SI values of 363 and 983, respectively. CM from TALL-104 cells induced by U937 (SI 717), Raji (SI 403), Daudi (SI 252), and HL60 (SI 88) cells also stimulated MO7E cell proliferation (Fig. 1 B). The stimulatory activity of all the CM raised in the presence of IL-2 was completely blocked by the combination of anti-GM-CSF and anti-IL-2 sera (not shown). In the presence of IL-2, the anti-CD2 and, especially, anti-CD3
659
LYMPHOKINE INDUCTION IN T-ALL CELLS TABLE 1
PRODUCTION OF IFN-'y AND TNF-a BY TALL-103/2 AND TALL-104 CELLS UPON STIMULATIONWITH mAb OR TUMOR CELLS TALL-103/2
TALL-104
No IL-2 Stimulus~
A. Tumor cells None K562 U937 Raji Daudi HL60 B. ImmobilizedmAb No mAb OKT3 B67.1 OKT3/B67.1
IL-2
No IL-2
IL~2
IFN-7
TNF-a
IFN-3~
TNF-a
IFN-'y
TNF-a
IFN-3~
TNF-a
