monoclonal lym-l antibody-dependent lysis of b-lymphoblastoid

Monoclonal Lym-l Antibody-Dependent Lysis of B-Lymphoblastoid Tumor Targets by Human Complement and Cytokine-Exposed Mononuclear and

Neutrophilic Polymorphonuclear Leukocytes By Lucian0 Ottonello, Paola Morone, Patrizia Dapino, and Franco Dallegri

Lym-1 is a murine IgG2a monoclonal antibody that recognizes a polymorphic variant of HLA-DR antigens on malignant B cells, with minimal cross-reactivity with normal tissues. Because it can be safely administered in vivo, a detailed knowledge of its ability to recruit and trigger the antitumor immune effector systems is required to optimize potential serotherapeutic approaches in B-lymphoma patients. By using Raji cells as a model of B-lymphoma targets, we found that Lym-l activates complement-mediated lysis efficiently. Moreover, Lym-l was capable of triggering the antibody-dependent cellular cytolysis (ADCC) by peripheral blood mononuclear cells (MNCs). On the contrary, it failed to trigger neutrophilic polymorphonuclear leukocyte (PMN)- mediated ADCC activity. In an attempt to enhance Lym-l ADCC by MNCs and PMNs, nine biologic response modifiers were tested. MNC-mediated Lym-l ADCC was significantly stimulated by interleukin-2 (IL-2) and unaffected by other

YM-l IS A MURINE IgG2a monoclonal antibody (MoAb) that recognizes a polymorphic variant of HLA-

DR antigens present on the surface of B-lymphoma cells and incapable of shedding or undergoing modulation after antibody binding.’ It has a relatively low reactivity with normal tissues and cells, including normal B lymphocytes.’ Moreover, radioimaging in vivo studies with ‘231-labeled Lym-l have shown antibody localization to sites of lympho- matous disease.’ Finally, by using Raji cells as a model of B-lymphoma target cells, Lym-l was found to trigger the cytolytic activity of complement3 and mononuclear cells (MNCS).~ Owing to these properties, Lym-l is presently con- sidered an attractive candidate for developing MoAb-based therapies of B lymphomas.

A preliminary clinical trial with Lym-l intravenous infusion, performed in 10 patients with refractory lymphoma, showed an evident reduction of lymph node size only in some cases.3 Although a number of factors can contribute to these partial response^,^ the inadequacy of host immune effector systems is likely to play a relevant role. To improve Lym-l antibody-based therapeutic approaches, it is therefore critical to understand whether cell-mediated cytolysis can be enhanced by biologic response modifiers. In this regard, two findings are promising, ie, the capacity of interleukin-2 (IL- 2) and y-interferon (7-IFN) to augment Lym-l antibody- dependent cytolysis by M N C S ~ . ~ and neutrophilic polymorphonuclear leukocytes (PMNS),~” respectively.

In the present study, we first examined the capacity of Lym- l to activate the lytic potential of the complement system, peripheral blood MNCs and PMNs. Then, in the attempt to increase the cell-mediated cytolytic activity, we tested a panel of nine biologic response modifiers having specific receptors on leukocytes.’”’

L

MATERIALS AND METHODS Culture medium and reagents. The following culture medium

was used: RPM1 1640 (Irvine Scientific, Santa Ana, CA) supple- mented with 10% heat-inactivated (56°C for 45 minutes) fetal calf serum (FCS; HyClone Europe Ltd, Cramlington, NE) and 2 mmol/

Blood, Vol 87, No 12 (June 151, 1996 pp 5171-5178

mediators, including y-interferon (y-IFN), tumor necrosis factor a (TNFa), and granulocyte-macrophage colony-stimulating factor (GM-CSF). On the other hand, PMN-mediated Lym-l ADCC was induced or significantly augmented by various cytokines, such as GM-CSF, TNFru, and yIFN, and chemotaxins, such as formyl peptides (FMLP), complement frag- ment C5a. and IL-8. Both MNC- and’ PMN-mediated ADCC was unaffected by granulocyte colony-stimulating factor (G- CSF) and insulin-like growth factor-I (IGF-l). Finally, only GM-CSF and TNFa augmented the number of PMNs actually engaged in the binding of Raji target cells. The findings pre- sented here, in particular those showing stimulatory activity of biologic response modifiers, may inspire new attempts for developing Lym-l antibody-based approaches to the therapy of B lymphomas. 0 1996 by The American Society of Hematology.

L glutamine (Irvine Scientific; RPMI-FCS). Hanks’ Balanced Salt Solution (HBSS) was from Irvine Scientific. Ficoll-Hypaque was purchased from Seromed (Berlin, Germany). Sodium chromate Cr 51 was from the Radiochemical Center (Amersham, UK). Triton X- 100, ethidium bromide, and fluorescein diacetate were purchased from Sigma Chemical CO (St Louis, MO). Heparin was obtained from Roche (Milano, Italy). Giemsa was purchased from Merck (Darmstadt, Germany). Human AB serum, pooled from three normal donors, was stored in aliquots at -80°C immediately after preparation or immediately after heat-inactivation (56°C for 45 minutes). Human CS-deficient serum was purchased from Sigma Chemical CO and stored in aliquots at -80°C. Monoclonal Lym-l antibody was a gift of Prof A.L. Epstein (Los Angeles, CA). A nonsense mouse IgG2a MoAb (Dako S.p.A., Milano, Italy) was used as an isotype control.

Cytokines and chemotaxins. Human recombinant tumor necrosis factor a (TNFa), human recombinant insulin-like growth factor-l (IGF-l), and human recombinant 72 amino-acids IL-8 were purchased from BioSource International (Camarillo, CA). Human recombinant y-IFN was obtained from Genzyme srl (Milano, Italy). Human recombinant C5a and N-formyl-met-leu-phe (FMLP) were purchased from Sigma Chemical Co. Human recombinant IL-2 was a gift of Prof F. Indiveri (University of Genova, Genova, Italy). Human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) and human recombinant granulocyte colony-stimulating factor (G-CSF) were kindly donated by Dr G. Guidi (Sandoz Italia, Milano, Italy). Each of these mediators was stored in aliquots

From the First Medical Clinic, Department of Internal Medicine,

Submitted October 26, 1995; accepted February 19, 1996. Supported by grants from MURST 40%-1993, 60%-1994, and

60%-1995 to F.D. Address reprint requests to Franco Dallegri, MD, Clinica Medica

I, Dipartimento di Medicina Interna, Viale Benedetto XV, n. 6, I - 16132 Genova, Italy.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact.

University of Genova, Genova, Italy.

0 1996 by The American Society of Hematology. 0006-4971/96/8712-0028$3.00/0

5171

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5172

at -80°C and was diluted with culture medium immediately before using.

Leukocyte preparation. Heparinized venous blood (10 U/mL heparin) was obtained from healthy volunteers (20 to 37 years old) after informed consent had been obtained. No donor had an infectious disease or was under medication at the time of and for 2 weeks before sampling. MNCs were isolated by centrifugation (400g for 30 minutes) on a Ficoll-Hypaque density gradient, washed with HBSS, and resuspended in RPMI-FCS, as previously described.l The resulting MNC preparations contained 70% to 87% lymphocytes, 13% to 29% monocytes, and less than 1% neutrophils or eosinophils, as determined by morphologic analysis of Giemsa- stained cytopreps. Cell viability was determined by ethidium bromide-fluorescein diacetate t e d 4 and was greater than 98%. Neutro- philic PMNs were prepared by dextran sedimentation, followed by centrifugation (400g for 30 minutes) on a Ficoll-Hypaque density gradient, as previously described." Contaminating erythrocytes were removed by hypothonic lysis." PMNs resuspended in RPMI-FCS were greater than 97% pure and greater than 98% viable, as determined by the assays described above.

Target cells. Lymphoblastoid Raji cells'' were used as targets in the cytolytic assays. The Raji cell line was grown in RPMI-FCS and subcultured every 3 days. The capacity of these cells to bind Lym-l antibody was measured by indirect immunofluorescence with flow cytometry using a rabbit antimouse IgG F(ab')z polyclonal antibody conjugated with fluorescein isothiocyanate (Dako). The Raji cell population contained 93.9% ? 0.8% positive cells (mean 2 I SD, n = 5). The mean fluorescence intensity of the cells was 23.2 t I . 1 (mean 2 1 SD, n = 5), with a standard deviation ranging from 30.0 to 35. I . For cytolytic assays, 4 X 10' Raji cells were labeled with 100 to 200 pCi sodium chromate Cr 51 by incubating for 1 hour at 37°C (final volume, 0.5 mL; medium, RPMI 1640 plus 5% FCS). After washing, labeled cells were resuspended in RPMI- FCS .

Cytolytic assays. Cytolytic activity of MNCs and PMNs was measured as described elsewhere in detail." Briefly, target cells (2 X IO4) were mixed with MNCs or PMNs at an effector:target ratio of 20: I , with and without Lym- 1 MoAb and cytokinedchemotaxins appropriately diluted in RPMI-FCS. The effector:target ratio of 20: 1 was chosen on the basis of preliminary experiments. To test the cytolytic activity of human complement, 2 X IO" cells were mixed with appropriate dilutions of human AB serum or heat-inactivated human AB serum or human CS-deficient serum in the presence or absence of Lym-l or the control IgG2a MoAb. The assays were performed in triplicate and in a final volume of I50 pL, using round- bottom microplates (Falcon, Becton-Dickinson Italia, Milano, Italy). After 14 hours of incubation in a humidified atmosphere of 95% air and 5% COz, the "Cr-release was determined in the cell-free supernatants. A series of experiments was also performed by prein- cubating (1 hour) either effector cells or target cells with biologic response modifiers. Some experiments were also performed using 20 hours of incubation. The percentage of cytolysis was calculated according to the formula 100 X (E - S)/(T - S), in which E is the cpm released in the presence of effector cells (or complement), T is the cpm released after lysing target cells with 5% Triton X-100, and S is the cpm spontaneously released by target cells incubated with medium alone (< 18%; with 4 exceptions: 20.4%, 2 1 . 4 7 ~ ~ 22. I %, and 25.7%).

Target cell binding assay. The assay was performed under conditions similar to those used for cytolytic assays. Briefly, 10' effector cells were mixed with 2 X lo4 Raji cells in the presence of Lym-l MoAb, with or without cytokines or chemotaxins. This effector:target ratio was chosen on the basis of preliminary experiments (higher effectorxarget ratios lead to cell overcrowding, therefore hampering the reading). Tests were performed in polypropylene 1.5 rnL capped

OTTONELLO ET AL

100 -

80 -

60 -

40 -

20 -

0 0 t. 5 10 15 20

Human serum (%) Fig 1. Dose-dependent induction of cytolysis by human serum in

the presence of 10 pg/mL Lym-l. "Cr-labeled Raji cells were at 2 x lo'. The incubation time was 14 hours. Results are expressed as the mean L 1 SD of five experiments.

Eppendorf-type tubes (Greiner GmbH, Frickenhausen, Germany) in a final volume of 150 pL. The tubes were centrifuged (501: for 5 minutes) and incubated for 20 minutes at 37°C in a humified atmosphere of 5% COz. After resuspension, the number of effector cells adherent to 1 0 0 target cells was determined by counting at least 200 Raji cells on Giemsa-stained cytopreps.

Statistical analysis. Results were expressed as the mean -+ 1 SD and/or as median with the 95% confidence interval. Statistical differences were analyzed by the Mann-Whitney test. Significance was accepted when P < .05.

RESULTS

When added to "Cr-labeled Raji cells in the presence of 10 pg/mL Lym-l, human serum induced a dose-dependent lysis of Raji target cells as measured by a 14-hour "Cr release assay (Fig 1). The magnitude of human serum-mediated lysis was also dependent on the Lym-l concentration (Fig 2) . These data suggest that (1) 10 pg/mL Lym-l and about 20% human serum are sufficient to induce the lysis of the majority of Raji target cells; and ( 2 ) relatively low concentrations of Lym-l (0.1 to 1 pg/mL), combined with 5% to 10% of human serum, are still capable of inducing substantial levels of cytolysis. Time-course experiments showed that the lysis reaches the plateau after 1 hour, ie, the serum-mediated Lym- 1 antibody-dependent cytolytic reac- tion is over at the end of I hour of incubation (data not shown). Heat-inactivated (56°C for 45 minutes) human serum, added to Raji cells in the presence of 10 pg/mL Lym- 1, was ineffective (percentage of cytolysis by 5% heat-inactivated serum, 0.7 t 0.6 [mean ? 1 SDI; n = 3). Similarly, CS-deficient human serum did not promote Lym- 1 antibody-




MODULATION OF LYM-l ADCC BY CYTOKINES 5173

100 r 80

60

40

20

t 0 1 4 rc 1 I

0.01 0.1 1 10 100

Lym-I ('g/mL) Fig 2. Effect of Lym-l concentration on the cytolysis induced by

human serum. Human serum concentrations were 10% (+ ),5% (m), and 1% (A). "Cr-labeled Raji cells were at 2 x lo4. The incubation time was 14 hours. The results shown are representative of two experiments.

dependent lysis (percentage of cytolysis by 5% CS-deficient serum in the presence of I O pg/mL Lym- I, 0.5 2 0.2 [mean ? 1 SDI; n = 5) . Finally, 20% human serum did not induce lysis of Raji cells in the presence of 10 pg/mL of control IgG2a MoAb (percentage of cytolysis, 0.4 ? 0.6 [mean 2 1 SDI; n = 3). Taken together, these results suggest that the lysis of Raji cells by human serum is due to Lym-l ability to trigger the cytolytic activity of the complement system.

Consistent with the resistance of Raji cells to natural killer (NK) MNCs caused negligible lysis (Fig 3). The addition of I O pg/mL Lym- 1 resulted in significant cytolysis, ie, MNCs appear to mediate Lym-l ADCC (Fig 3 and its legend). On the other hand, PMNs were incapable of mediat- ing significant spontaneous and Lym-l antibody-dependent cytolysis (Fig 3 and its legend). As shown in Fig 3, there was intersubject variability in MNC-mediated ADCC, apparently unrelated to the number of monocytes in the MNC popula- tions used (data not shown). Moreover, PMNs from relatively few individuals were found to exert ADCC (Fig 3). Nevertheless, repetitive testing of same donors at various times indicated that the majority had consistent profiles of both MNC- and PMN-mediated ADCC, ie, high responders usually displayed high activity, whereas low responders gen- erally had low activity (data not shown). As summarized in Fig 4, the addition of 200 U IL-2 at the beginning of the assay significantly (legend to Fig 4) augmented MNC-mediated Lym-l ADCC. On the other hand, when added at the beginning of the assay in absence of Lym-l, IL-2 did not significantly (P = .420) affect the negligible activity of MNCs (percentage of cytolysis by MNCs, 0.9 2 1.1 [mean

2 I SDI; n = 5; median, 0.6; confidence interval 95%, -0.5 to 2.4; percentage of cytolysis by MNCs plus 200 U IL-2, 2.7 2 2.7 [mean 2 1 SDI; n = 5 ; median, 2.9; confidence interval 95%. -0.7 to 6.1). Therefore, under the present conditions, IL-2 stimulates MNC-mediated Lym- 1 ADCC without affecting the antibody-independent activity, ie, without inducing lymphokine-activated killer (LAK) cell activity. Stimulation of MNC-mediated Lym-l ADCC could also be detected using Lym-l at the concentration of 1 pg/mL in-

a 0

t

k 0 10

Lym-l (pg/mL)

* 2ot E Q)

a, 2

a

a

4 l 0 0 10

Lym-l (pg/mL) Fig 3. MNC- and PMN-mediated cytolysis in the absence or pres-

ence of 10 pg/mL Lym-l. 5'Cr-labeled Raji cells were at 2 x lo4. The MNC Raji cell ratio and PMN:Raji cell ratio were 20:l. The incubation time was 14 hours. MNC-mediated cytolysis was 1.5 2 1.9 (mean ? l SD, n = 10). with a median of 0.85 (confidence interval 95%, 0.1 t o 2.91, in the absence of Lym-l and 20.7 ? 10.5 (mean 2 1 SD, n = 351, with a median of 19.9 (confidence interval 95%. 17.1 to 24.3). in the presence of Lym-l. MNC-mediated cytolysis in the absence of Lym- 1 versus that observed in the presence of Lym-l was P < .0001. PMN- mediated cytolysis was 0.9 ? 1.3 (mean 1 SD, n = 13). with a median of 0.0 (confidence interval 95%. 0.1 t o 1.71, in the absence of Lym-l and 3.0 ? 5.6 (mean l SD, n = 42). with a median of 0.7 (confidence interval 95%. 1.3 to 4.81, in the presence of Lym-l. PMN- mediated cytolysis in the absence of Lym-l versus that observed in the presence of Lym-l was P = .1309.




5174

60 - 50 - 40 - 30 - 20 - 10 -

0 200

lL-2 (U/mL) Fig 4. Effect of 11-2 on MNC-mediated Lym-l antibodydependent

"Cr-labeled Raji cells (2 x 10'). The MNC: Raji cell ratio was 201. Lym-l was at 10 pg/mL. The incubation time was 14 hours. The cytolysis was 21.9 2 12.0 (mean 2 1 SD, n = 19). with a median of 19.9 (confidence interval 95%. 16.1 to 27.71, in the absence of IL-2 and 31.5 & 14.0 (mean 2 1 SD, n = 19). with a median of 32.4 (confidence interval S%, 24.7 to 38.31, in the presence of IL-2. Cytolysis in the absence versus that in the presence of IL-2 was P = .0343.

stead of 10 pg/mL and 100 U but not 50 U/mL IL-2 instead of 200 U (data not shown). Finally, a panel of other cytokines or biologic mediators were tested for their potential to increase MNC-mediated Lym-l ADCC (Table 1). Although each of these agents was found to enhance ADCC by MNCs from one or more donor, none of them also had significant activity (Table 1) after preincubations with MNCs or target cells (data not shown). Because yIFN and GM-CSF have been previously claimed to enhance Lym-l -dependent MNC cytolysis in a 20-hour assay,6.I6 parallel experiments

OTTONELLO ET AL

were performed using 14-and 20-hour incubation periods. Both 100 U/mL yIFN and 1 ng/mL GM-CSF also did not significantly stimulate MNC-mediated Lym-l ADCC in the 20-hour assay (percentage of stimulation of MNC ADCC by

during 14- and 20-hour incubation periods, respectively; percentage of stimulation of MNC ADCC by GM-CSF, 13.0 ? 6.1 [ P = ,2221 and 9.5 +- 8.9 [P = S471 during 14- and 20- hour incubation periods, respectively [mean -+ 1 SDI; n =

5). As shown in Figs 5 and 6, two cytokines (GM-CSF and

TNFa) and two chemotaxins (FMLP and C5a) induced or enhanced PMN-mediated Lym-l ADCC significantly (leg- ends to Figs 5 and 6). When added to PMNs plus Raji cells in the absence of Lym-l or to Raji cells incubated with Lym- 1 in absence of PMNs, they had no effect on the 5'Cr release from target cells. Stimulation of PMN-mediated Lym-l ADCC by GM-CSF and FMLP was also detected using Lym- 1 concentrations less than than 10 pg/mL (Fig 7). The same phenomenon could be observed using TNFw and C5a (data not shown). As for MNC-mediated Lym-l ADCC, other agents were tested for their ability to increase the target lysis by PMNs (Table 2). Among them, yIFN and IL-8 significantly augmented PMN-mediated Lym- 1 ADCC (Ta- ble 2), but the magnitude of the target lysis was relatively low as compared with that observed using GM-CSF, TNFa, FMLP, and C5a. Other cytokines were completely ineffective, with low intersubject variability (Table 2), and were unable to stimulate PMN-mediated Lym- 1 ADCC even after preincubation with PMNs or Raji cells (data not shown). As summarized in Fig 8, GM-CSF and TNFa were found to significantly increase PMN binding to Raji cells in the presence of Lym-l, whereas other biologic response modifiers were ineffective.

./-IFN, 13.2 ? 2.6 [P = .222] and 14.9 ? 7.9 [P = ,4201

DISCUSSION

The present study, performed using Raji cells as a model of B-lymphoma targets, shows that (1) Lym-l antibody activates the cytolytic sequence of human complement system efficiently; (2) Lym-l triggers the ADCC activity of MNCs through a process susceptible of significant enhancement by

Table 1. Effect of Various Mediators on Lym-l Antibody-Depemdent Cytolysis by MNCs

Percentage of Cytolysis'

Mediatort Mediator Present Mediator Absent

Type Dose Mean 2 1 SD Median Confidence Interval 95% Mean 2 1 SD Median Confidence Interval 95% n PValue

GM-CSF 1 ng/mL 19.3 2 7.2 21.1 14.4-24.1 17.8 It 5.3 18.6 14.2-21.4 11 .519

G-CSF l ng/mL 24.5 -c 12.3 27.7 13.0-35.9 20.9 ? 11.1 20.4 10.5-31.2 7 ,535

yIFN 100 U/mL 24.1 -c 8.3 20.8 15.3-32.9 24.2 -t 5.4 23.4 18.5-39.9 6 ,818

TNFa 1 ng/mL 24.5 2 14.2 23.4 13.6-35.5 22.6 z 10.5 23.9 14.5-30.7 9 .730 IGF-1 100 ng/rnL 27.5 5 10.4 29.4 19.4-35.5 22.6 2 10.5 23.9 14.5-30.7 9 .340 IL-8 l prnol/L 20.2 lr 10.9 21.2 11.1-29.4 19.8 2 10.6 20.2 10.8-28.7 8 ,720 FMLP 1 pmol/L 23.7 lr 11.5 24.0 14.8-32.5 22.6 ? 10.5 23.9 14.5-30.7 9 ,863 C5a 1 ImollL 28.2 ? 16.5 18.5 13.4-38.9 22.6 ? 10.5 23.9 14.5-30.7 9 1.000

* Percentage of cytolysis at 20:l MNCRaji cell ratio. Lyrn-l concentration was 10 pg/mL. Incubation time was 14 hours. t When added to MNCs plus Raji cells in absence of Lyrn-l or mixed with Raji cells plus Lyrn-l, none of the agents was found to induce Raji

cell lysis.





'I U)

g 40 c

0 ~~~~~~

1

GM-CSF (ngImL)

0 1 TNFa (ng/mL)

Fig 5. Effect of GM-CSF and TNFa on PM-mediated Lym-l antibody-dependent cytolysis. "Cr-labeled Raji cells were at 2 x lo4. The PMN: Raji cell ratio was 201. Lym-l was at 10 Fg/mL. The incubation time was 14 hours. (A1 The cytolysis was 1.8 f 2.6 (mean f 1 SD, n = 231, with a median of 0.5 (confidence interval 9546, 0.6 to 2.91, in the absence of GM-CSF and 19.2 f 10.1 (mean f 1 SD, n = 231, with a median of 20.0 (confidence interval 95%. 14.8 to 23.61, in the presence of GM-CSF. Cytolysis in the absence of GM-CSF versus that in the presence of GM-CSF was P < .0001. (B) The cytolysis was 5.1 f 7.6 (mean f 1 SD, n = 19). with a median of 2.6 (confidence interval 95%. 1.4 to 8.71, in the absence of TNFa and 17.1 f 10.8 (mean f 1 SD. n = 19). with a median of 14.5 (confidence interval 95%. 11.8 to 22.31, in the presence of TNFa. Cytolysis in the absence of TNFa versus that in the presence of TNFm was P < ,0001. When added to PMNs and Raji cells in the absence of Lym-l or mixed with Raji cells plus Lym-l, both GM-CSF and TNFa had no effect.

IL-2; and (3) although per se ineffective, Lym-l induces ADCC by PMNs in the presence of GM-CSF, TNFq FMLP, and C5a. Therefore, Lym-l alone or in combination with biologic response modifiers is capable of directing and activating some of the major immune cytolytic systems towards B-lymphoblastoid tumor cells.

Previous experiments showed that complement lyses Raji cells presensitized with high doses of L ~ m - 1 . ~ The present findings extend this observation, showing that high levels of

target lysis can be induced by 5% to 20% human serum in presence of Lym-l concentrations achievable in vivo.3 In agreement with previous Lym-l was also found to trigger MNC-mediated lysis, an event enhanced by IL-2. This finding, coupled with the incapacity of IL-2 to induce LAK activity in the present setting, suggests that the signals delivered by Lym-l and IL-2 converge and synergize to amplify the ADCC activity of MNCs. None of the other cytokines herein tested, including yIFN and GM-CSF, was

* c Q) e a!

30

15

0

A A

- I

0 l

FMLP (pmol/L)

0 1

C5a (pmol/L) Fig 6. Effect of FMLP and C5a on PMN-mediated Lym-l antibody-

dependent cytolysis. "Cr-labeled Raji cells were at 2 x lo4. The PMN- Raji cell ratio was 201. Lym-1 was at 10 pg/mL. The incubation time was 14 hours. (AI The cytolysis was 4.1 f 7.0 (mean f 1 SD, n = 241, with a median of 1.1 (confidence interval 95%. 1.1 to 7.11, in the absence of FMLP and 17.5 f 9.4 (mean f l SD, n = 24). with a median of 17.2 (confidence interval 95%. 13.5 to 21.51, in the presence of FMLP. Cytolysis in the absence of FMLP versus that in the presence of FMLP was P < .OOOl. (B) The cytolysis was 5.3 f 8.0 (mean f 1 SD, n = 17). with a median of 2.6 (confidence interval 95%. 1.1 to 9.41, in the absence of C5a and 11.6 2 9.0 (mean f 1 SD, n = 17). with a median of 11.4 (confidence interval 95%. 7.0 to 16.3) in the presence of C5a. Cytolysis in the absence of C5a versus that in the presence of C5a was P = .0103. When added to PMNs and Raji cells in the absence of Lym-l or mixed with Raji cells plus Lym-l, both FMLP and C5a had no effect.




5176 OTTONELLO ET AL

m% 40

8 30 - 0

GM-CSF : r F 20.

k 10- Q, 0

4 0; I

0.01 0.1 1 10 100 Lym- l (pg/mL)

20 Q) c, 5 10 4

FMLP

Nil I

GM-CSF I I *

FMLP l i

Y-IFN I /L -8 I c5a l - i

I I I I

0 40 80 120 160

Raji cell-bound PMNs/100 Raji cells

Fig 8. Effect of biologic response modifiers on the PMN binding to Raji cells in the presence of 10 mg/mL Lym-l. GM-CSF was at 1 nglml; FMLP was at 1 pmol/L; TNFa was at 1 nglml; ylFN was at 100 U/mL; IL-8 was at 1 pmolll; and C5a was at 1 pmol/L. The resub (number of Raji cell-bound PMNsl100 Raji cells) are shown W the mean f 1 SD, n = 5. * P = .M11 (GM-CSF v Nil); **p = . O M 1 (TNFa v Nil). Other mediators versus Nil are not significant.

found to enhance Lym-l ADCC by MNCs, even during a 20-hour assay and after 1 hour of preincubation with either effector or target cells. Therefore, our data do not confirm

.r I I

0.01 0.1 1 10 100 ""

I

the findings of other investigators showing that y-IFN6 and GM-CSFL6 stimulate Lym-l ADCC by MNCs in most indi-

Fig 7. Effect of Lym-l concentration on the enhancement of PMN- viduals. mediated cytolysis by GM-CSF and FMLP. "Cr-labeled Raj1 cells were As regards PMN-mediated ADCC, it is well-known that at 2 x lo'. The PMN:Raji cell ratio was 201. The incubetion time was these p~gocytes lyse lymphoid cells, including ~ ~ j i cells, 14 hours. The concentrations of GM-CSF were 1 nglmL 101, 0.1 ng/ in the presence of rabbit antitarget antibodies.~7 Despite this mL (m), and 0.01 ng/mL (A), and those of FMLP were 1 pmollL (01,

GM-CSF and FMLP was 0.0 at each Lvm-l concentration. The results vestigators,6 PMNs failed to exert Lym-l ADCC. Never- 0.1 pmol/L (m), and 0.01 pmol/L (A). The cytolysis in the absence of ability and consistent with the results obtained by Other in-

~~ ~~

shown are representative of two experiments. theleis, two cytokines (GM-CSF and TkFa) and two chemotaxins (FMLP and C5a), per se ineffective in absence of Lym-l, were found to induce Lym-l ADCC by PMNs.

Table 2. Effect of Various Mediators on Lym-l Antibody-Dependent Cytolysis by PMNS

Percentage of Cytolysis*

Mediatort Mediator Present Mediator Absent

Type Dose

G-CSF 1 ng/mL 6.0 t 7.9 2.5 0.3-11.7 7.6 It 9.6 3.0 0.6-14.0 10 .853 7-IFN 100 U/mL 6.4 t 3.8 6.6 3.9-2.8 1.4 t 2.0 0.1 0.1-2.8 12 .0005 IL-2 1 ng/mL 2.2 t 2.0 1.9 0.7-3.6 2.2 t 2.1 2.5 0.8-3.6 11 .S47

IGF-1 100 ng/mL 6.2 t 6.3 3.6 1.6-10.7 7.6 t 9.6 3.0 0.6-14.5 10 ,911 IL-8 1 pmol/L 4.1 t 3.1 4.4 2.5-5.7 1.9 t 2.1 1 .o 0.8-2.9 17 ,035

Mean i- 1 SD Median Confidence Interval 95% Mean ? 1 SD Median Confidence Interval 95% n f'value

Percentage of cytolysis at 20:l PMN:Raji cell ratio. Lym-l concentration was 10 pg/mL. Incubation time was 14 hours. t When added to PMNs plus Raji cells in absence of Lym-l or mixed with Raji cells plus Lym-l, none of the agents was found to induce Raji

cell lysis.





Moreover, y-IFN and IL-8 displayed significant but very low PMN stimulatory activity. As for the IL-2 enhancement of Lym-l ADCC by MNCs, these results suggest the inter- vention of a synergistic interaction between Lym-l and each of the aforementioned biologic mediators. However, at present, we have no hint as to the detailed biochemical processing whereby these distinct mediators exert the same stimulatory action. Nevertheless, the enhancement of PMN ADCC by GM-CSF and TNFa is associated with an augmentation of the number of target cell-bound effectors, whereas other mediators (FMLP, C5a, y-IFN, and L-8) stimulate ADCC of bound PMNs without affecting the effector-target conjugate formation. Finally, it is of note that, whereas the results obtained with y-IFN are confirmatory,6 those observed with GM-CSF, TNFq FMLP, and C5a are novel. In fact, to our knowledge, only GM-CSF was previously found to stimulate the MoAb-dependent tumoricidal activity of PMNs, using melanoma, neuroblastoma, or certain colorectal carcinoma target

The ability of Lym-l shown here to mediate complement- dependent cytolysis efficiently, coupled with its particular reactivity for lymphoma cells,' suggests its possible use for the purging of harvested bone marrow before reinfusion. On the other hand, preliminary clinical studies with Lym-l intravenous infusion in patients with refractory lymphomas have shown low response rates? suggesting that the in vivo activation of complement- and MNC-mediated cytolysis by Lym-l is relatively inefficient as compared with in vitro findings. Nevertheless, the administration of Lym-l might be effective in patients with a low tumor burden. As shown in biopsy specimens, Lym-l can trigger a sort of inflammatory or immune response at tumor sites, at least in a subset of lymphoma patients3 Similarly, certain antitumor and complement-activating MoAbs have been found to induce tumor-destructing inflammatory reactions in melanoma patients." Therefore, although radiolabeled or toxin-conjugated Lym-l may also be effective,23 the possibility of aug- menting the activity of immune cells by using biologic response modifiers appears to be a reasonable option to improve Lym-l antitumor effects. Taking into account the intersubject variation of lymphoma cell ability to bind Lym- 1,' the combination of the antibody with IL-2 to increase MNC-ADCC might specifically target and potentially eradi- cate small numbers of residual tumor cells in selected patients. Moreover, the capacity of certain cytokines (GM-CSF and TNFa) and chemotaxins (FLMP and C5a) to trigger PMN-mediated Lym- l ADCC raises potentially attractive possibilities to develop new approaches to the serotherapy of lymphomas. Although the infusion of cytokines or other mediators has its own set of problems, the known toxicity of intravenous administration of TNFa,"." the ability of IL- 2 to promote the production of TNFcI,~~ and the expected unwanted effects of FMLP and C5a26 might be reduced by conjugation of these molecules with the antibody. As far as GM-CSF is concerned, it has been recently shown that this cytokine potentiates Lym-l ADCC by macrophages." This finding and the present data, coupled with the ability of GM- CSF to increase monocyte-macrophage production28 and PMN production and s u r ~ i v a 1 , 2 ~ ~ ~ ~ render the cytokine the

best candidate for inducing phagocytes to express their Lym- 1 -dependent antitumor potential. Consistent with this possibility, the administration of an antitumor MoAb together with GM-CSF has yielded encouraging clinical responses in patients with colorectal car~inoma.~'

REFERENCES 1. Epstein AL, Mader RJ, Winter JN, Stathopoulos E, Chen FM,

Parker J W , Taylor CR: Two new monoclonal antibodies, Lym-l and Lym-2, reactive with human B-lymphocytes and derived tumors, with immunodiagnostic and immunotherapeutic potential. Cancer Res 47:830, 1987

2. Epstein AL, Zimmer AM, Spies SM: Radioimmunodetection of human B-cell lymphomas with a radiolabeled tumor specific monoclonal antibody, Lym-l, in Cavalli F, Bonadonna G, Rozenc- weig M (eds): Malignant Lymphomas and Hodgkin Disease: Experi- mental and Therapeutic Advances. Boston, MA, Martinus Nijhoff, 1985, p 569

3. Hu E, Epstein AL, Naeve CS, Gill I, Martin S, Sherrod A, Nichols P, Chen D, Mazumder A, Levine AM: A phase la clinical trial of Lym-l monoclonal antibody serotherapy in patients with refractory B cell malignancies. Hematol Oncol 7:155, 1989

4. Gill I, Agah R, Hu E, Mazumder A: Synergistic antitumor effects of interleukin 2 and the monoclonal Lym-l against human Burkitt lymphoma cells in vitro and in vivo. Cancer Res 49:5377, 1989

5. Biddle WC, Pancook J, Goldrosen M, Han T, Foon KA, Vaickus L: Antibody-dependent, cell-mediated cytotoxicity by an anti-class I1 murine monoclonal antibody: Effect of recombinant interleukin-2 on human effector cell lysis of human B-cell tumors. Cancer Res 50:2991, 1990

6. Vaickus L, Biddle W, Cemerlic D, Foon KA: Interferon gamma augments Lym-l-dependent, granulocyte-mediated tumor cell lysis. Blood 75:2408, 1990

7. Cemerlic D, Dadey B, Han T, Vaickus L: Cytokine influence on killing of fresh chronic lymphocytic leukemia cells by human leukocytes. Blood 77:2707, 1991

8. Aggarwal BB, Pocsik E: Cytokines: From clone to clinic. Arch Biochem Biophys 292:335, 1992

9. Fu YK, Arkins S, Wang BS, Kelley KW: A novel role of growth hormone and insulin-like growth factor-l. Priming neutrophils for superoxide anion secretion. J Immunol 146:1602, 1991

10. Djen JY, Liu JH, Wei S, Rui H, Pearson CA, Leonard WJ, Blanchard DK: Function associated with the IL-2 receptor-B on human neutrophils. Mechanism of activation of antifungal activity against candida albicans by IL-2. J Immunol 150:960, 1993

1 1. Dinarello CA: Role of interleukin-l and tumor necrosis factor in systemic responses to infection and inflammation, in Gallin JI, Goldstein IM, Snyderman R (eds): Inflammation: Basic Principles and Clinical Correlates. New York, NY, Raven, 1992, p 211

12. Gerard C, Gerard NP: The pro-inflammatory seven-trans- membrane segment receptors of the leukocytes. Curr Opinion Immu- no1 6:140, 1994

13. Dallegri F, Patrone F, Frumento G, Ballestrero A, Sacchetti C: Down-regulation of K cell activity by neutrophils. Blood 65:571, 1985

14. Dankberg F, Persidsky M: A test of granulocyte membrane integrity and phagocytic function. Cryobiology 13:430, 1976

15. Safrit JT, Bonavida B: Hierarchy of in vitro and resistance of tumor cells to cytotoxic effector cells, cytokines, drugs and toxins. Cancer Immunol Immunother 34:321, 1992

16. Cairo MS, Wang J, Plunkett JM, Norvitch M, Van de Ven C: GM-CSF and IL-3 significantly enhance the antibody-dependent




5178 OTTONELLO ET AL

cytotoxicity (ADCC) of the Lym-l antibody against B-cell malig- nancy. Proc Am Assoc Cancer Res 32:261, 1991 (abstr)

17. Dallegri F, Patrone F, Frumento G, Sacchetti C: Antibody- dependent killing of tumor cells by polymorphonuclear leukocytes. Involvement of oxidative and nonoxidative mechanisms. J Natl Can- cer Inst 73:331, 1984

18. Kurshner BH, Cheung NKV: GM-CSF enhances 3F8 monoclonal antibody-dependent cellular cytotoxicity against human melanoma and neuroblastoma. Blood 75:1936, 1989

19. Baldwin GC, Chung GY, Kaslander C, Esmail T, Reisfeld RA, Golde DW: Colony-stimulating factor enhancement of myeloid effector cell cytotoxicity towards neuroectodermal tumour cells. Br J Haematol 83:545, 1993

20. Ragnhammar P, Frodin JE, Trotta PP, Mellstedt H: Cytotoxic- ity of white blood cells activated by granulocyte-colony-stimulating factor, granulocytehacrophage-colony-stimulating factor and macrophage-colony-stimulating factor against tumor cells in the presence of various monoclonal antibodies. Cancer Immunol Immunother 39:254, 1994

21. Harmenberg J, Hoglund M, Hellstrom-Lindberg E: G- and GM-CSF in oncology and oncological haematology. Eur J Haematol 52:1, 1994

22. Vadhan-Raj S, Cordon-Cardo C, Carswell E, Mintzer D, Dantis L, Duteau C, Templeton MA, Oettgen HF, Old LJ, Houghton AN: Phase I trial of a mouse monoclonal antibody against GD3 ganglioside in patients with melanoma: Induction of inflammatory responses at tumor sites. J Clin Oncol 6:1636, 1988

23. Grossbard ML, Press OW, Appelbaum FR, Bemstein ID, Nadler LM: Monoclonal antibody-based therapies of leukemia and lymphoma. Blood 805363, 1992

24. Schell-Frederick E, Tepass T, Lorscheidt G, Pfreundsche M, Schaadt M, Diedl V: Effects of recombinant tumor necrosis factor (rhuTNFa) on human neutrophils and monocytes: In vitro, ex vivo and in vivo. Eur J Haematol 43:286, 1989

25. Heslop HE, Gottlieb DJ, Bianchi ACM, Maerger A, Pren- tice HHG, Metha AB, Hoffbrand AV, Brenner MK: In vivo induction of gamma interferon and tumor necrosis factor by interleukin- 2 infusion following intensive chemotherapy or autologous bone marrow transplantation for hematologic malignancies. Blood 77:1374, 1989

26. Styrt B: The role of phagocytes in non-infectious diseases, in Klempner MS, Styrt B, Ho J (eds): Phagocytes and Diseases. Dor- drecht, The Netherlands, Kluwer, 1989, p 145

27. Charak BS, Agah R, Mazumder A: Granulocyte-macrophage colony-stimulating factor-induced antibody-dependent cellular cytotoxicity in bone marrow macrophages: Application in bone marrow transplantation. Blood 81:3474, 1993

28. Golde DW, Baldwin GC: Myeloid growth factors, in Gallin JI, Goldstein IM, Suyderman R (eds): Inflammation: Basic Principles and Clinical Correlates. New York, NY, Raven, 1992, p 291

29. Brach MA, de Vos S, Gruss HJ, Herrmann F: Prolongation of survival of human polymorphonuclear neutrophils by granulocyte- macrophage colony-stimulating factor is caused by inhibition of pro- grammed cell death. Blood 80:2920, 1992

30. Ragnhammar P, Fagerberg J, Frodin JE, Hjelm AL. Linde- malm C, Magnusson I, Masucci C, Mellstedt H: Effect of monoclonal antibody 17-IA and GM-CSF in patients with advanced colorectal carcinoma: Long-lasting, complete remission can be induced. Int J Cancer 53:751, 1993




1996 87: 5171-5178

L Ottonello, P Morone, P Dapino and F Dallegri mononuclear and neutrophilic polymorphonuclear leukocytestumor targets by human complement and cytokinine-exposed Monoclonal Lym-1 antibody-dependent lysis of B-lymphoblastoid

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