A human T-T-cell hybridoma-derived lymphocyte chemoattractant factor

CELLULAR

IMMUNOLOGY

98,4 1l-42 1 ( 1986)

A Human T-T-Cell Hybridoma-Derived Lymphocyte Chemoattractant Factor’*2 ARTHUR

C. THEODORE,~ DAVID M. CENTER, WILLIAM W. CRUIKSHANK,

AND DENNIS J. BEERS Pulmonary Center, Evans Memorial Department of Clinical Research, Boston University School of Medicine, Boston, Massachusetts 02118 Received July 15, 1985; accepted November 19, I985

Human T-T hybridomas were developed as a strategy for obtaining lympbokines that alter Tlymphocyte motility. Mitogen-stimulated human T lymphocytes were fused with cells of the human CEM lymphoma line and the supematantsderived from thesefusion products were assessed for chemoattractant activity in a modified Boyden chamber assay.Supematants from hybridoma 4 1B2 enhanced lymphocyte migration to 198 f 13%(mean ? SEM) of control. Characterization by Sephadex G-100 molecular sieve chromatography revealed a single peak of chemoattractant activity correspondingto a molecular weight (MW) of 56,000.This activity eluted from a Sephadex QAE anion-exchange column at 4-6 mS. Subsequent isoelectric focusing in sucroserevealed an isoelectric point of 9.0-9.2. Fractions with activity after sequential molecular sieve and anionexchange chromatography were concentrated, radiolabeled with ‘251,and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography revealed a band which corresponded to a MW of 14,000 (representing four similar monomeric chains) and to the region from which chemoattractant activity could be detected in eluates from slices of unstained gels run in parallel. The biological activity of this hybridoma-derived lymphocyte chemoattractant was abolished by treatment with trypsin and neuraminidase but was unaffected by heating to 56°C. We conclude that certain human T-T-cell hybridomas constitutively elaborate a lymphocyte chemoattractant that appears to be physicochemically identical to a previously described human lymphokine, lymphocyte chemoattractant factor. 0 1986 Academic press, Inc.

INTRODUCTION

Human lymphocytes elaborate lymphokines which are involved in the modulation of immune responses (l-3). Of these factors, several lymphocyte chemoattractants may be important in the recruitment and immobilization of nonsensitized effector T lymphocytes at sites of inflammation (4-9). In vitro, stimulation of human histamine type-2 (HZ) receptor-bearing T lymphocytes results in the production of a selective T’ Presentedin part at the 68th Annual Meeting of the Federation of American Societies for Experimental Biology, St. Louis, MO., June 1984. * Supported in part by U.S. Public Health Service Grants HL 29544 and HL 32802 and a grant from the American Lung Association. 3Dr. Theodore is the recipient of National Serice Research Award HL 06975. ’ Dr. Beer is an E. L. Trudeau Scholar of the American Lung Association and the recipient of Clinical Investigator Award HL 00898 from the National Institutes of Health. To whom all correspondenceshould be addressed. 411 0008-8749186 $3.00 Copyright 0 1986 by Academic F-res. Inc. All rights of reproduction in any form reserved.

412

THEODORE ET AL.

lymphocyte chemoattractant factor (LCF), a cationic sialoprotein with a molecular weight (MW) of 56,000 which is composed of four 14,000-Da biologically active monomers (8- 11). In addition, histamine type- 1 (H,) receptor-bearing T lymphocytes elaborate two inhibitors of T-cell migration. These lymphocyte migration inhibitory factors (LyMIFs) are cationic proteins with MW of 75,000 and 35,000 (11, 12). Unfortunately, stimulated T cells produce these molecules in less than nanomolar amounts, making further identification of primary structure and mechanisms of action difficult. In an attempt to generate larger quantities of these lymphocyte chemoattractants, we established human T-T-cell hybridomas. Several of these hybridomas were capable of the constitutive elaboration of LCF. MATERIALS AND METHODS Medium 199 (M 199; Whittaker Bioproducts, Walkersville, Md.); RMPI 1640 (Flow Laboratories, McLean, Va.); fetal bovine serum (FBS), Hepes buffer, penicillin streptomycin solution (GIBCO Laboratories, Grand Island, N.Y.); bovine serum albumin (BSA; Miles Laboratory, Inc., Elkhart, Ind.); Sartorius nitrocellulose 8-pm micropore filters (Sartorius Filters, Inc., Cherry Hill, N.J.); Neuroprobe chemotactic chambers (Neuroprobe Inc., Cabin John, Md.), ampholytes 7 to 10 (Bio-Rad Laboratories, Rockville Center, N.Y.); Ficoll-Paque, Sephadex G-100, Sephadex QAE (Pharmacia Fine Chemicals, Pitscataway, N.J.); Sprague-Dawley rats (Charles River, Boston, Mass.); polyethylene glycol MW 1500, neuraminidase, emetine hydrochloride, mitomycin C, actinomycin D, 2-mercaptoethanol, phytohemagglutinin (PHA) (Sigma Chemical Co., St. Louis, MO.); immobilized TPCK-trypsin (Pierce Chemical, Rockford, Ill.); OKT3-FITC monoclonal antibody (Ortho Diagnostics, Raritan, N.J.); [3H]thymidine, ‘25I (New England Nuclear Corp. Boston, Mass.) were obtained from manufacturers and suppliers. Development of stable human T-T hybridomas. Human peripheral blood T lymphocytes (13, 14) suspended in RMPI 1640 supplemented with 2 mA4 L-glutamine, 100 U/ml penicillin, 100 pg/ml streptomycin, 10% FBS, and 5 X 10T5M 2-mercaptoethanol (final medium) were blast transformed by incubating lo6 cells/ml with 1 pg/ml PHA in a humidified 5% CO2 atmosphere. After 48 hr, the cells were washed in serum-free medium and incubated with 0.1 M N-acetyl-D-galactosamine in RPM1 1640 at 37°C for 20 min in order to remove cell-bound PHA. CEM cells, obtained from the American-Type Culture Collection, growing in logarithmic phase were harvested and prepared for fusion by a variation of the method of Kobayashi et al. (15). The CEM cells were incubated in medium containing 5 X 10e5A4 emetine hychochloride and 0.25 pg/ml actinomycin D at 37°C for 2 hr in a humidified 5% CO2 atmosphere. These concentrations of emetine and actinomycin D inhibited proliferation of the CEM cells completely. Following incubation, the cells were harvested and washed four times with 10 mM phosphate-buffered saline (PBS), pH 7.2. The T cells were washed twice with serum-free RPM1 1640 and copelleted with the CEM cells at a ratio of 3:2 (T cells:CEM cells). Fusions were carried out using the method of Irigoyen et al. (16). One-half milliliter of prewarmed (37’C) 46% polyethylene glycol 1540 (PEG), 15%dimethyl sulfoxide, and 5 pg/ml poly+arginine was added down the side of the tube over 45 set and the pellet resuspendedgently. The mixture was immediately centrifuged for 3 min at 58Og.Without removing PEG, the cells were resuspended in 10 ml of prewarmed serum-free medium and centrifuged at 250g for 10 min. After fusion the cells were washed twice and resuspended in final medium. Feeder cells (CEM cells incubated with 50 pg/ml mitomycin C for 2 hr at 37°C) were added to

HYBRIDOMA-DERIVED

LYMPHOCYTE

CHEMOATTRACTANT

FACTOR

413

the fusion suspension at a ratio of 4 X 1O5feeder cells: lo6 CEM cells and placed in microtiter plates at a concentration of 4 X IO’ cells/well. The cultures were maintained at 37°C in a humidified 5% CO2 atmosphere and were fed three times per week. These fusions have resulted in T-T-hybridoma growth within 4 to 6 weeks with fusion frequencies between 23 and 62%. Control wells containing unfused cells showed no evidence of growth. Karyotype analysis of the CEM lymphoma and established hybridomas was performed by routine staining techniques ( 15). The CEM lymphoma cells had a diploid (46) number of chromosomes, but the T-T-cell fusions all had greater than diploid number of chromosomes per cell. Lymphocyte migration. Lymphocyte migration was assessedby employing a modification of a Boyden chamber technique (S- 12) utilizing rat splenic T lymphocytes as the responding cell pool. It has been shown previously that the migratory response of rat splenocytes to the human lymphocyte chemoattractant lymphokines is identical to that of human T cells (8-12). To determine the effect of an experimental sample on lymphocyte migration, 50 ~1 of a 10 X lo6 cells/ml suspension of rat splenic T lymphocytes in medium 199 supplemented with 25 mM Hepes buffer (100 U/ml penicillin and 100 pg/ml streptomycin; M 199-HPS) enriched with 0.4% BSA was placed in the upper compartments of 48-well microchemotaxis chambers separated from 30 ~1 of final medium or PBS, pH 7.4 (controls), or experimental samplesby S-pm nitrocellulose filters. Lymphocyte migration experiments were carried out for 3 hr at 37°C in a humidified 5% COZ atmosphere. The filters were fixed, stained, dehydrated, and mounted using standard histological techniques. Lymphocyte migration was quantified by counting the total number of cells migrating beyond a fixed distance using lo-pm intervals in five highpower fields (hpf) in duplicate micropore filters. This distance was arbitrarily set at a point where lo-20 cells per hpf had migrated under control conditions. Results were calculated as mean cells/hpf + SD. Mean data from similar experiments underwent analyses of variance to determine statistical significance. A P value of less than 0.05 was considered significant. In all figures, experimental data were expressedas mean percentage f SD of migration under control conditions. Chromatography. Crude supernatant (15 ml) from human T-T-cell hybridomas was harvested and concentrated, and 1 ml of the concentrate was applied to a 1.5 X 90cm Sephadex G-100 gel filtration column and eluted with PBS, pH 7.4. Fractions (2 ml) were collected in glass tubes and 30 ~1 of each fraction was placed in the lower wells of duplicate chemotactic chambers and assessedfor effects on lymphocyte migration as described previously. Aliquots of PBS, pH 7.4, from the void volume of the corresponding column served as controls. Fractions with chemoattractant activity were pooled, dialyzed against distilled HZ0 to remove excesssalt, and subjected to Sephadex QAE anion-exchange chromatography. Samples with chemoattractant activity after molecular sieve chromatography were applied to a 1O-ml SephadexQAE exchange column, equilibrated with 0.0035 M PBS, pH 7.8, and eluted with a linear loo-ml salt gradient to 1 M NaCl at 4°C. Fractions (4 ml) were collected and each fraction’s ionicity was determined using a Copenhagen conductivity meter. The fractions were then dialyzed separately in PBS, pH 7.4, and 30 ~1of each fraction was evaluated for chemoattractant activity. In some experiments, fractions containing lymphocyte chemoattractant activity after sequential molecular sieve and anion-exchange chromatography were subjected to trypsin degradation or neuraminidase treatment.

414

THEODORE ET AL.

Isoelectric focusing in sucrose was performed with a Buchler apparatus. Sucrose gradients (lo-40% 20 ml) were generated utilizing a Buchler gradient maker and a constant flow pump containing ampholytes in the 7-10 pH range. Electrophoresis wasperformed at constant current for 2 hr. Fractions (1 ml) were eluted with a peristaltic pump, assessedfor pH, and dialyzed against PBS, pH 7.4, for 24 hr at 4°C to remove ampholytes and sucrose. Samples (30 ~1) were placed in the lower wells of microchemotaxis chambers and assessedfor lymphocyte chemoattractant activity. Sequential molecular sieve and anion-exchange chromatography and isoelectric focusing were performed on three separate aliquots of supernatant from hybridoma 41B2. The elution profiles of lymphocyte chemoattractant activity were consistent and reproducible from all of the aliquots. Under Results, these data are presented as a representative chromatogram. Lymphokine iodination. Radioiodination was performed utilizing Iodo-Beads. Two Iodo-Beads were incubated at 25°C with 200 ~1of PBS, pH 7.4, for 5 min and dried. ‘25I (1 mCi) was then added to the Iodo-Beads in 100 ~1of PBS, pH 7.4. Concentrated lymphokine preparation (200 ~1, derived from the concentration of 45 ml of crude supernatant which underwent sequential molecular sieve and anion-exchange chromatography) was applied to washed radiolabeled beads and allowed to incubate for 15 min at 25°C. The reaction was terminated by aspirating off the supernatant containing iodinated lymphokine. Electrophoresis. Iodinated lymphokine was then prepared for electrophoresis in sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Samples were mixed with a nonreducing buffer consisting of 24% glycerol. 18-40% SDS (w/v), 58% 0.5 M Tris, pH 6.8, at a ratio of 40% sample:60%buffer. This solution (30 ~1)was applied to lanes of a 14% SDS-PAGE in a Bio-Rad slab gel apparatus. Molecular weight standards mixed with bromophenol blue were placed in the end lanes. Electrophoresis was performed at 30 mA at constant current until the tracking dye had reached the bottom of the gel. The gel was then removed, stained with silver stain, and dried in a solution containing 45% methanol, 1% glycerol, and 10% acetic acid. The gel was dried onto filter paper for 3 hr at 80°C in a Bio-Rad gel drier. When the gel was dry, L251-labeled proteins were detected by autoradiography. Duplicate samples in adjacent lanes were removed and sliced in l-cm sections prior to staining. Sections were individually eluted in PBS at 4’C for 5 days and then assayedfor chemoattractant activity. Suppressor assay. Crude supernatants derived from hybridomas were assayedfor suppressor activity (17). One million human peripheral blood mononuclear cells (PBMC), suspended in final medium supplemented with 15%human serum (control) or in supernatants derived from hybridomas supplemented with 15% human serum, were stimulated with 0.5 p/ml PHA in microtiter plates (2 X 10’ cells/well) and incubated in a humidified 5% CO2 atmosphere for 3 days. Eighteen hours prior to culture termination, [3H]thymidine (1 &i/well) was added. The incorporation of [3H]thymidine into cellular DNA was determined by harvesting the cultures on glasswool filter paper with a MASH II harvester. Radioactivity was detected by liquid scintillation counting. The mean number of counts per minute (&SD) was determined from quadruplicate wells. The variability of the assaywas less than 10%. Percentage suppression was calculated using the formula % suppression = 1.O -

(mean cpm hybridoma supernatant) x 100. (mean cpm final medium)

HYBRIDOMA-DERIVED

LYMPHOCYTE

CHEMOATTRACTANT

FACTOR

415

RESULTS IdentiJication

of Hybridoma-Derived

Lymphocyte

Chemoattractant

Activity

Supernatants harvested from 8-week-old hybridoma cell cultures were evaluated for the presenceof lymphocyte chemoattractant activity. Of the 55 hybridoma cultures screened,8 contained lymphocyte chemoattractant activity. Supernatant derived from culture 4 1B2 caused the greatest increase in lymphocyte migration ( 198 f 13%,mean -t SEM, n = 3) and was investigated in further detail. Supernatants derived from the parent CEM cells did not affect T-cell migration (98 +- 7%). The T lymphocytes which elaborate LCF bear both the T8 antigen and H2 receptors ( 10, 18). At least one other human lymphokine (histamine-induced suppressor factor, HSF) has been demonstrated to be a product of T8+, H2 recpetor-bearing lymphocytes ( 17, 19). In order to determine if both lymphokine activities were elaborated from the same hybridomas, we assayedthe supernatants derived from several hybridomas for both T-lymphocyte chemoattractant and suppressor activities. As shown in Table 1, several hybridomas which elaborated lymphocyte chemoattractant activity did not produce any suppressoractivity while three hybridomas which elaborated a suppressor activity did not produce lymphocyte chemoattractant activity. We did not characterize the suppressor activity further. Physicochemcial tant Activity

Characterization

of Hybridoma-Derived

Lymphocyte Chemoattrac-

Supernatants from hybridoma 41B2 were harvested, pooled, concentrated by lyophilization, and fractionated by Sephadex G- 100 molecular sieve chromatography. A single peak of lymphocyte migration enhancement was observed in fractions corresponding to a MW of 55,000-60,000 (Fig. 1). Lymphocyte migration inhibitory activity was not present in any of the fractions. Concentrated final medium containing EBS (control) fractionated by identical means showed no chemoattractant activity (Fig. 1). Fractions with lymphocyte chemoattractant activity from molecular sieve

TABLE 1 Chemoattractant and SuppressorActivities in Hybridoma Supematants

Hybridoma CEM (control) 41B2 41B, 41B4 41c3 41c, 41DZ

Chemoattractant activity” (%I 98k 198 f 107+ 173+ 106k 91f 152+

3 13c 5 3’ 5 6 4c

SuppressorActivity b @I 2+2 -7 + 4 20 + 4d -2 Lk5 25 + 3d 23 t 2d 9+4

‘All results are expressed as percentage of control migration + SEM for three experiments. Control migration was 17.83 + 2.32 cells/hpf (mean k SD, n = 10). ‘All results are expressedas percentage suppression + SEM for three experiments. ’ Denotes migratory responsesdifferent from control, P < 0.01. d Denotes suppression of mitogen-stimulated peripheral blood lymphocyte proliferation different from control, P < 0.0 1.

416

THEODORE ET AL.

-

70

-

J.

1

1

60 / 4/

40

60

60

100

% Bed Volume

FIG. 1. Sephadex G-100 gel chromatogram of crude supematant derived from hybridoma 41Br (0) and from final medium alone (0). Every other fraction was assayedfor ability to alter the random migration of rat splenic T cells. The rat T-cell migratory responsesare expressedon the ordinate as percentageof migration in control buffer alone, where control migration was 18.0 + 2.62 cells per hpf (mean + SD, n = 10). Data points that are above the horizontal (100%) line represent increasesin migration compared to that of buffer control, while those that are below represent migratory responsesthat are lessthan control. Molecular weight markers indicated are blue dextran (BD, MW 2 X 106) bovine serum albumin (BSA, MW 68,000),ovalbumin (OA; MW 43,000), and vitamin BIZ (Brr; MW 12,000). A single peak of chemoattractant activity eluted at a MW of 55,000-60,000 Da. No lymphocyte migration inhibitory activity was observed. *P < 0.0I.

chromatography were pooled, dialyzed against distilled H20, and subjected to QAE anion-exchange chromatography. Lymphocyte chemoattractant activity eluted early in the salt gradient at an ionicity of 4.0-6.0 mS (Fig. 2). The total protein concentration of the crude supernatant derived from hybridoma 4 1Bz, determined by the standard Bio-Rad protein assay, was 3 mg/ml. After sequential molecular sieve and anionexchange chromatography, the pooled fractions with lymphocyte chemoattractant activity had a total protein concentration of 200 pg/ml. Isoelectric focusing of chemoattractant activity after sequential molecular sieve and anion-exchange chromatography revealed a single area of activity in fractions corresponding to a pI of 9.09.2 (Fig. 3). The nature of the biological activity of hybridoma-derived lymphocyte chemoattractant activity obtained after sequential chromatography was further characterized by enzymatic degradation and heat treatment (Table 2). Lymphokine was incubated at 20°C with TPCK-Trypsin beads at an equivalent trypsin concentration of 10e7M. After 30 min the lymphokine was eluted with PBS and assessedfor effects on lym-

HYBRIDQMA-DERIVED

LYMPHOCYTE

CHEMOATTRACTANT

417

FACTOR

200 20 160

19

160

16 17

170

I6 160

15

150

14 13

140

I2 130

II

120

IO 0

110

6 100

7

QO

6 5

60

4 70

3 2

60

1 123458

i

a

0

10

11

12

13

14

15

16

17

16

19

20

Fraction

FIG. 2. Sephadex QAE anion-exchange chromatogram of pooled lymphocyte chemoattractant activity from molecular sieve chromatography. Rat splenic T-lymphocyte migratory responsesare expressedon the ordinate as percentage of migration in control buffer alone, where control was 18.2 + 2.39 cells per hpf (mean f SD, n = 10).Conductance (0) of individual fractions, measuredat 4”C, is expressedon the opposite ordinate as mS. Peak chemoattmctant activity eluted early in the salt gradient, corresponding to an ionicity of 4.0-6.0 mS. *P < 0.01.

phocyte migration. After trypsinization chemoattractant activity was abolished ( 184 + 13% vs 107 + 5%). Lymphokine incubated with lo-’ M neuraminidase for 30 min similarly abolished its migratory enhancement effect ( 184 + 13%vs 109 f 3%). Heating the lymphokine to 56°C for 30 min had no significant effect on its biological activity (184 -t 13% vs 176 + 7%). SDS-PAGE

and Autoradiography

of Hybridoma-Derived

Lymphokine

Hybridoma 4 1B2-derived lymphokine activity obtained after sequential molecular sieve and anion-exchange chromatography was radiolabeled with lz51and subjected to SDS-PAGE and autoradiography. Radiolabeling of this lymphokine preparation did not result in loss of biological activity (data not shown). As control, supematant derived from the CEM lymphoma line underwent identical sequential molecular sieve and anion-exchange chromatography. Fractions from this column control eluting at 4-6 mS were iodinated and subjected to SDS-PAGE and autoradiography. As shown in Fig. 4, autoradiography of the experimental sample derived from hybridoma 4 1BZ revealed a band corresponding to a MW of 14,000. Two other bands were visualized of slightly higher MW. None of these three bands was present in autoradiograms from

THEODORE ET AL.

418 200 100 160 170

0.8

160

10.4

160

10.0

140

D.6

130 a.2 120 8.6 110 8.4

I

100 B.0

f,

00 7.6 .BC

7.2

7c

6.6 6.4 0.0

I 12345670

0

10

11

12

13

14

16

16

17

18

10

20

Fraction

FIG. 3. Isoelectric focusing of lymphocyte chemoattractant activity from sequential molecular sieve and anion-exchangechromatography. Rat splenic T-lymphocyte migratory responsesare expressedon the ordinate as percentage of migration in control buffer alone where control was 19.1 + 2.02 cells per hpf (mean + SD, n = 10). The pH (0) of individual fractions, measured at 4°C is expressed on the opposite ordinate. A single peak of chemoattractant activity was demonstrated in fractions with a pH of 9.0-9.2. *P < 0.01.

the CEM control. A large band representing contaminating albumin can be seen at MW 60,000-70,000 in both the hybridoma and control lanes. Eluates of parallel unstained gels from hybridoma 4 1B2 sliced in 1-cm segmentsrevealed chemoattractant TABLE 2 Effect of Enzymes or Heat on Hybridoma-Derived Lymphokine Activity

Buffer 4lB,-LCF

Control

Tryp-‘a

Neur-”

100+ 4d 184 + 13’

107 +- 5 107 f 5

105 ?I 3 109 + 3

Heat 56°C’ ND’ 176 + 7.0f

’ An aliquot of the test hybridoma lymphokine was incubated with TPCK-Trypsin beads( lo-’ M trypsin) for 30 min at 37°C eluted from the column, and then placed in the lower wells of microchemotaxis chambers. Aliquots of PBS incubated with TPCK-Trypsin Beads served as controls. b Neuraminidase at 10m7M was incubated with test lymphokine or PBS for 30 min at 37°C in the lower wells of microchemotaxis chambers before assay. Neuraminidase ( 10e7M) had no effect on lymphocyte migration. ‘An aliquot of the test lymphokine was heated to 56°C for 30 min, and then placed in the lower wells of microchemotaxis chambers. d PBS (buffer control) was placed in the lower wells of microchemotaxis chambers. All results are expressed as percentage of control migration + SEM for three experiments. Control migration was 14.0 + 1.0 cells per hpf (mean rt_SD, n = 10). ’ Not done. Denotes migratory responsesdifferent from control, P < 0.01.

HYBRIDOMA-DERIVED Molecular

LYMPHOCYTE Weight

A

CHEMOATTRACTANT B

FACTOR

419

Slices 1

94,000.

. 2 I

68,000.

3 I 4 43,000-

I

30,000.

I

5 6 . 7

21,ooom

I 8

14,000-

I 9

FIG. 4. Autoradiograph of SDS-PAGE of lymphocyte chemoattractant obtained from sequential molecular sieve and anion-exchangechromatography aher labeling with “‘1 (lane B). Lane A representsautoradiography of SDS-PAGE of LZ51-labeled final media (control) after sequential molecular sieve and anion-exchange chromatography. Numbers on the right correspond to slices of unstained gels run in parallel which were eluted and assayedfor lymphocyte chemoattractant activity.

activity only in the section corresponding to a MW of 13,000-17,500 (Fig. 5). The total protein concentration eluted from this segment of the gel was 1 pg/ml. Since we could not be certain that all this protein represents biologically active LCF, we could not determine the specific activity of this lymphokine preparation. No activity was observed in eluates of the corresponding sliced portions from CEM control (data not shown). These characteristics of hybridoma-derived LCF are identical to those of human blood T-lymphocyte-derived LCF (8 10). DISCUSSION In humans, the production of LCF has been demonstrated to be restricted to H2 receptor-bearing TV lymphocytes (10, 18). This cell population elaborates LCF in response to stimulation with mitogen or histamine (S-10, 18). However, because of the small quantities of lymphokine that are ordinarily generated by human cells under the usual culture conditions, it has been difficult to obtain enough material to study lymphokine-target cell receptor interaction and mechanisms of lymphokine-induced cell activation. We have demonstrated that a human T-T-cell hybridoma constructed by fusing PHA-activated peripheral blood T cells with emetine and actinomycin D-treated CEM cells elaborated a lymphocyte chemoattractant identical to human peripheral blood T-cell-derived LCF. Hybrid formation was documented by demonstrating greater than diploid numbers of chromosomes in fused cells after 8 weeks of culture. Emetine- and actinomycin D-treated CEM cells as well as PHA-activated peripheral blood T cells did not survive past 14 days in culture. Because the screening for lymphocyte chemoattractant activity was performed at least 8 weeks after fusions were done, the possibility that LCF was produced directly by unfused T cells could be excluded. The failure of the CEM parent line to elaborate LCF, and the long-term production of LCF over 8 months provided further evidence that the production of lymphocyte chemoattractant activity was limited to a T-T-cell hybridoma.

THEODORE ET AL.

420

200 IS0 130 170 150 150 140 130 120 110 100 60 60 70 60

1

23456788

Slice FIG. 5. Lymphocyte chemoattractant activity eluted from slices of unstained parallel gels from Fig. 4. Lymphocyte chemoattractant activity is expressedon the ordinate as percentage of control migration where control was 13.0 f 2.1 cells/hpf (mean + SD, n = 10). *P c: 0.01.

The hybridoma 4 1Bz-derived T-cell chemoattractant activity eluted from a Sephadex G-100 column in a region corresponding to a MW of 55,000-60,000 (Fig. 1). This elution pattern is identical to that previously reported for lymphocyte-derived LCF (8- 10). Application of this lymphocyte chemoattractant activity to a Sephadex QAE anion-exchange column revealed that it eluted early in the salt gradient corresponding to an ionicity of 4.0-6.0 mS (Fig. 2). Isoelectric focusing of this material revealed a single area of activity corresponding to a p1 of 9.0-9.2 (Fig. 3). Hybridoma-derived LCF obtained after sequential column chromatography and isoelectric focusing was treated with a number of enzymes. The biological activity of this material was sensitive to both trypsin and neuraminidase treatment, suggestingthat the hybridoma-derived LCF is a glycoprotein (Table 2). This pattern of physicochemical characteristics is identical to that previously reported for T-lymphocyte-derived LCF (8- 10). As noted on SDS-PAGE, interaction of hybridoma-derived LCF with SDS resulted in the appearance of lymphocyte chemoattractant activity at a MW corresponding to 13,000-17,500 (Figs. 4 and 5). This suggeststhat hybridoma-derived LCF, like that derived from activated blood T cells, was elaborated as an aggregatedtetramer with a MW of 56,000 which disassociated to biologically active monomers (MW 14,000) during SDS-PAGE (8,9). The three other radiolabeled bands seen on SDS-PAGE of chromatographed supematant from hybridoma 4 1B2demonstratesthat this preparation of LCF is not pure. The large MW band appearing at MW 60,000-70,000 seen on

HYBRIDOMA-DERIVED

LYMPHOCYTE

CHEMOATTRACTANT

FACTOR

421

electrophoresis of supernatant from both the CEM lymphoma line and hybridoma 4 1B2 after sequential molecular sieveand anion-exchange chromatography represented albumin that was a component of the culture medium. No lymphocyte chemoattractant activity was observed from corresponding slices of the unstained gel run in parallel. There were two other bands seenbetween MW 17,500 and 2 1,000 on autoradiography of SDS-PAGE of 41B2 hybridoma-derived supernatant but not that of CEM-derived supernatant. The eluates of the slices of the unstained parallel gel corresponding to MW 17,500-21,000 did not contain lymphocyte chemoattractant activity while the slice corresponding to MW 13,000-17,500 containing two bands did possesschemoattractant activity. In addition to providing a chemoattractant lymphokine in greater quantities for further study of its mechanism of action, thesehybridomas provide evidence supporting the functional diversity of T-lymphocyte subpopulations. The T lymphocytes which elaborate LCF and HSF bear both T8 antigen and H2 receptors (10, 17- 19). Our data suggesta functional heterogeneity of the T8+ H2 receptor-bearing lymphocyte population since hybrid 41B2 elaborated LCF but not any suppressor activity. However, other hybridomas were able to produce suppressor activity but failed to produce lymphocyte chemoattractant activity. Thus, the T8+ H2 receptor-bearing lymphocyte subset is a heterogeneous population with respect to lymphokine production. ACKNOWLEDGMENTS We thank Dr. Lanny J. Rosenwasserfor thoughtful discussionsand Laurie M. Beardsley and Lisa Derby for their excellent assistancein the preparation of this manuscript.

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