HUMAN B CELL LINES EXPRESS THE INTERFERON GAMMA GENE Mark A. Dayton, Thomas J. Knobloch, David Benjamin Interferon y (IFNy) production has been attributed exclusively to activated T cells and NK cells. We sought to determine whether human B cells express IFN-y. We studied 28 B cell lines including Epstein-Barr virus (EBV)+ normal lymphoblastoid B cell lines (N = 7), EBV+ B cell lines derived from patients with Burkitt’s lymphoma with (N = 6) or without AIDS (N = S), as well as seven EBV- B cell lines. All cell lines were studied by reverse transcription-polymerase chain reaction (RT-PCR). We detected constitutive expression of IFN-y in every B cell line. The tumor promoters PMA and teleocidin appeared to enhance this IFN-y expression in nearly every B cell line. The 517 bp amplicons spanning the entire protein coding region of the IFN-y mRNA from three representative lines were sequenced, definitively establishing that B cell IFN-y is identical to IFN-y from activated T cells and is not altered by derivation of the B cell lines from AIDS patients or by EBV status. Detection of IFNy in the entire panel of EBV+ and EBV- cell lines suggests that the IFN-7 gene is broadly expressed by human B cells. Our data imply that human B cells can be activated to produce IFN-y, further enmeshing B cells in the dynamics of immunoregulation.

Until recently, interferon y (IFN-y) was believed to be produced exclusively by activated T cells and natural killer (NK) cells (reviewed in refs 1, 2). Mounting evidence suggests, however, that the expression of IFN-?I is not so severely restricted. Using monoclonal antibodies to several epitopes of rat IFNy, Olsson et al. detected IFN-y-like immunoreactivity in skeletal muscle fibers,3 axotomized motor neurons,4 and nerves of the central and peripheral nervous systems.5 Based on biological and immunological assays, Naumovski et al.6 described IFN-y secretion from the SUP-HDl cell line derived from a patient with Hodgkin’s Disease. Furthermore, IFN-y release

From the Department of Internal Medicine, Division of Hematology and Oncology, The Ohio State University Comprehensive Cancer Center/Arthur G. James Cancer Hospital and Research Institute, Columbus, Ohio 43210, USA. Address correspondence and requests for reprints to: Mark Dayton, MD, PhD, Division of Hematology and Oncology, James Cancer Hospital and Research Institute, Room 1232, 300 West 10th Ave., The Ohio State University, Columbus, Ohio, 43210-1228, USA. Received 28 February 1992; revised and accepted for publication 21 August 1992 0 1992 Academic Press Limited 104s4666/921060454+07 $08.00/O KEY WORDS: B cell lines/gene gamma/lymphokine/RT-PCR

expression/Interferon

has been described from the two Epstein-Barr virus positive (EBV+) human B-lymphoblastoid cell lines GM3299 and GM4408 after induction by elevated temperatures,7 and from the EBV+ Burkitt’s lymphoma cell line JLP(C) upon stimulation by the tumor promoter teleocidin.8 Pang et al.9 have recently demonstrated that JLP(C) and another EBV+ Burkitt’s lymphoma cell line, PA682BM-1, express mRNA that hybridizes to probes from a human IFN-y cDNA and secrete a protein neutralized by anti-IFN-y antibodies. Given the hybridization data, the RIA measurements, and the activity neutralization by anti-IFN-y antibodies,s,g it seems very likely that B cells indeed produce authentic IFN-?I. However, it is possible that this B cell interferon and IFN-y are related molecules which share antigenic epitopes but are not identical. As variants of B cell IL-l and tumor necrosis factor p (TNF-p) have been recently described,lO,ll we elected to study the expression of B cell IFN-y at the molecular level to definitively establish its identity. In this report, we conclusively demonstrate that B cell IFN-y is identical to IFN-?/ produced by T cells and NK cells throughout the entire protein coding region of the mRNA.12 This was accomplished by cloning and sequencing the IFN-y message from several representative B cell lines. Furthermore, we ascertained that the expression of IFN--y in B cell lines is not an aberration uniq.ue to a few lines, but is a CYTOKINE,

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B cell interferon

more generalized phenomenon of EBV+ and EBV- cell lines.

seen in a wide panel

RESULTS Expression

of IFN-y

mRNA by RT-PCR Analysis

Twenty-eight B cell lines were analyzed for IFN-?/ mRNA expression using reverse transcription-polymerase chain reaction (RT-PCR). The study included tumor B cell lines derived from patients with undifferentiated lymphoma of Burkitt’s and nonBurkitt’s types (N = 22) and normal lymphoblastoid B cell lines (N = 7) (Table 1). Since phorbol myristate acetate (PMA) has been shown to induce IFN-y in several T cell clone&2 all cell lines were also activated for 24 hours with 50 ng PMA/ml or 10 ng teleocidin/ml. Teleocidin is known to share similar biological activities with PMA and

TABLE

1.

IFN-7

mFtNA expression

in human

gamma

B cell lines.

Normal lymphoblastoid cell lines

EBV status

Burkitt’s Burkitt’s Burkitt’s Burkitt’s Burkitt’s Burkitt’s

Positive Positive Positive Positive Positive Positive

Burkitt’s Burkitt’s Undiff.

PMA 50 ngml

PA682BM-1 PA682BM-2 PA682PE-1 PA682PE-2 PA682PB AS283

++ + ++ + ++ +

++ ++ + ++ ++ ++

+ ++ + ++ ++ ++

Positive Positive Positive

DW6 KK124 JLP(C)

+ ++ +++

+ ++ +++

++ + +++

Negative Negative Negative Negative Negative Negative Negative

MC116 JD38 JD39 EW36 DS179 CA46 ST486

+ + ++ ++ ++ ++ +

+ + ++ ++ + ++ ++

+ ++ ++ ++ ++ ++ ++

non-AABCL

Burkitt’s Burkitt’s Burkitt’s Burkitt’s Burkitt’s

Positive Positive Positive Positive Positive

AK778 HRI+2 RAJI DAUDI NAMALVA

++ + + + ++

+++ + ++ ++ ++

++ ++ ++ ++ ++

non-AABCL

Cord blood Cord blood Cord blood Mononucleosis Mononucleosis Mononucleosis Peripheral blood

Positive Positive Positive Positive Positive Positive Positive

CB23 CB33 CB34 IM1178 IM-B182 IM-El82 PACB-PE

++ + ++ + + + ++

++ ++ ++ + + + ++

++ + ++ + + + ++

Undiff. Undiff. Undiff. Undiff. Undiff. Burkitt’s Burkitt’s

lymphoma lymphoma lymphoma lymphoma lymphoma lymphoma

Cell line

Analysis

Teleocidin 10 nghl

AABCL

Source

RT-PCR

Constitutive

non-AABCL

African Burkitt’s lymphoma cell lines

/ 455

to bind to the same specific cellular receptors as PMA, although structurally it is different.13 PMA may mimic cytokine activity in biological assays. In contrast, no such interference was observed with teleocidin.14 Total cellular RNA was extracted from control (constitutive), teleocidin-activated and PMAactivated cells, Reverse transcription for the RT-PCR was accomplished using random primers. The specific PCR primers bracketed the entire protein coding region of the mRNA for IFN-y. Primer 1 included the ATG translation initiation codon and primer 2 complemented the 3’ end of the protein coding region including the TAA translation termination codon. Amplified fragments were detected by ethidium bromide staining after electrophoresis through 2% agarose gels. Representative amplicons are shown in Fig. 1. Constitutive expression of substantial amounts of IFN-y mRNA has been found in 14 of the 28 B cell lines examined, and detectable amplification was seen

IFN-y

American lymphoma cell lines

expression

The constitutive expression of IFN-?/ mRNA detected by RT-PCR in 28 B cell lines is summarized. IFN-7 gene expression has been detected in all B cell lines by RT-PCR analysis. Constitutive expression of substantial amounts of IFN-y mRNA has been found in 14 of the 28 B cell lines examined, and detectable amplification was seen in the other 14 B cell lines. IFN-7 expression was stimulated by teleocidin in most cell lines. OKT3 activated, IL-2 stimulated CD8+ T cells expressed an amount of IFN-7 mRNA that was equivalent to those seen in several B cell lines. Neutrophils did not express IFN-y mRNA in apreciable amounts. + = Positive; ++ = strongly positive; +++ = very strongly positive. AABCL = AIDS associated B cell lines; Burkitt’s = Burkitt’s lymphoma; undiff. lymphoma = high grade undifferentiated lymphoma.

456 I Dayton

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et al.

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Figure 517 bp sentative

1992: 454-460)

1. RT-PCR analysis of amplicons from reprehuman B cell lines.

Constitutive IFN-y mRNA expression from representative cell lines of each category of Table 1 is demonstrated. Lanes 1 and 7 contain a 100 bp ladder (Gibco BRL, the bold band just above the IFN-7 amplicons is 600 bp); lane 2, PA682BM-1 (4 ~1); lane 3, JLP(C) (4 ~1); lane 4, CA46 (20 ~1); lane 5, AK778 (4 ~1); lane 6, IM-B182 (8 ~1); lane 8, no RNA for RT (20 ~1); lane 9, no cDNA for PCR (20 ~1); and lane 10, RNA but no cDNA for PCR (20 ~1). Volumes are the amount of a 100 ~1 PCR reaction loaded into the gel. The 517 bp fragment can be seen in all of the lines, but in none of the controls. Amplified fragments were detected by ethidium bromide staining after TAE electrophoresis through 2% agarose gels.

517 bp +

in the.other 14 B cell lines (Table 1). IFN-y expression was easily detected in every B cell line following stimulation by teleocidin. The levels of EN-y mRNA determined from the B cell lines JLP(C) and AK778 were greater than those found in OKT3 activated, IL-2 stimulated, CD8+ T cells. Several other B cell lines expressed levels of IFN-y mRNA similar to that in T cells (Table 1). Furthermore, IFN-y mRNA expression was not found in neutrophils under

conditions which allowed easy detection of IFN-y in the B cell lines with the lowest levels of expression (Fig. 2). The PCR controls were uniformly negative and included samples with no RNA for reverse transcription, no reverse transcribed cDNA for PCR, PCR of RNA that had not been reverse transcribed, and PCR with no Tuq DNA polymerase. Northern blots of RNA from the two B cell lines PA682BM-

A lanes 517

B lanes

I

2

3

4

5

6

7

8

9

IO

II

12

13

I4

15

I6

I7

18

bp+

I

Figure

2

3

2.

The effect

4

5

of teleocidin

6

and PMA

7

8

on IFN-y

9

mRNA

IO

II

12

I3

14

15

16

expression.

Stimulated IFN-y mRNA expression in representative B cell lines from each category in Table 1 was assessed at 24 hours following stimulation with or without PMA (50 rig/ml) or with teleocidin (10 ngiml). Lane 1, CD8+, OKT3 activated, IL-2 stimulated T cells; lanes 2-4, PA682PB; lanes 5-7, AK778; lane 8, no RNA control; lanes P-11, DW6; lanes 12-14, DS179; lanes 15-17, IM-B182; lane 18, neutrophils. Lanes 2, 5, 9, 12 and 15 were unstimulated, constitutive levels of expression; lanes 3, 6, 10, 13 and 16 were stimulated with teleocidin; lanes 4, 7, 11, 14 and 17 were stimulated with PMA. Amplicons were detected by incorporating [&zP]-dCTP into the product during PCR as described in the methods, TBE electrophoresis through 2% agarose gels and electro-blotting to a nylon membrane. (A) Four minute film exposure showing high levels of expression in CD8+ T cells, PA682PB, and AK778 cell lines. (B) Forty-minute exposure showing lower levels of expression in DW6, DS179, and IM-B182. Neutrophils did not express IFN-y in appreciable amounts.

I7

18

B cell interferon gammaexpression/ 457 1 and JLP(C), demonstrated the expected 1.3 kb message hybridizing to a T-cell-derived IFN-y probe (ref. 9, unpublished data). Finally, using a similar protocol, we have previously shown that some of these B cell lines produce IL-lo, while others show no message for IL-lo,15 indicating that the detection of 1FN-r expression is not merely an artifact of the exquisite sensitivity of PCR.

patients (AIDS-Associated B cell lines - AABCL); the EBV+ cell line JLP(C) represents American Burkitt’s lymphoma derived from a non-AIDS patient (non-AABCL); and the EBV- cell line MC116 represents an undifferentiated lymphoma of Burkitt’s type obtained from an American patient. All three B cell lines showed the same IFN-71 sequence, which was also identical to the IFN-y sequence described previously.12

Stimulation of IFN- y mRNA Expression The cell line JLP(C) was studied by RT-PCR for IFN-y mRNA expression following stimulation with or without teleocidin or PMA. Both teleocidin and PMA induced the expression of IFN-y mRNA in this cell line with maximum effects being seen at about 9-24 hours after beginning stimulation (Fig. 3). IFN-)I expression in JLP(C) activated with these tumor promoters was comparable to or even exceeded that from OKT3 activated, IL-2 stimulated CD8+ T cells.

DISCUSSION We demonstrate the expression of IFN-)I mRNA in a wide panel of tumor B cell lines derived from patients with AIDS and Burkitt’s lymphoma, American Burkitt’s and African Burkitt’s lymphoma, as well as normal lymphoblastoid B cell lines. Our data show that all of these B cell lines constitutively express IFN-y mRNA and suggest that these B cell lines can further augment the expression of IFN-y. That this IFN-y message expression is real is supported by the following: (i) the primers used for the PCR amplifications were specifically designed to hybridize to authentic IFN-y and the amplicons were of the expected size; (ii) all of the appropriate controls were negative; (iii) previous studies have documented the release of an IFN-y-immunoreactive molecule from two of these B cell line@; (iv) we have previously studied these B cell lines for IL-10 mRNA expression using essentially the same RT-PCR protocol.15 In the case of IL-lo, several of these B cell lines had no detectable mRNA expression, whereas others demonstrated amplification similar to what we find for IFN-y . We have shown that the sequence of B cell IFN-?/ is identical to IFN-7 produced by T cells and NK cells throughout the entire protein coding region of

Cloning and SequencingB Cell IFN-y RT-PCR products from amplifications using primers 1 and 2 were directionally cloned into pBluescript II for sequencing. Plasmids containing the appropriate size inserts, as determined by electrophoresis in 2% agarose minigels after cutting with Pvu II, were completely sequenced. The SK and KS primers for pBluescript II were used to obtain sequence information for both strands of the insert from double-stranded plasmid DNA. We cloned and sequenced both strands of the 517 bp IFN-y PCR fragments spanning the entire IFN-y protein coding region from three representataive B cell lines: PA682BM-1, JLP(C) and MC116. The EBV+ cell line PA682BM-1 was selected since it represents American Burkitt’s lymphoma in AIDS

Constitutive

Hours

I

Figure

5

3.

9

Tie

24

course

Teleocidin IO rig/ml 48

72

of stimulated

I

IFN--y

5

9

expression

PMA 50 rig/ml 24

from

48

72

I

5

9

24

48

72

C

IL-2 CD8+ T ceils

--

216

the cell line JLP(C).

JLP(C) cells were stimulated for the indicated times (1, 5, 9, 24,48 and 72 hours) in the presence or absence of the tumor promoters teleocidin (10 rig/ml) or PMA (50 rig/ml). Lanes ld were unstimulated, constitutive levels of expression; lanes 7-12 were stimulated with teleocidin; lanes 13-18 were stimulated with PMA; lane 19 was a ‘no RNA’ control; lane 20 was from CD8+, OKT3 activated, IL-2 stimulated T cells. The 517 bp fragment can be seen in all of the lines and the CD8+ cells but not in the ‘no RNA’ control (C). Amplicons were detected by incorporating [a-s*P]-dCTP into the product during PCR as described in the methods, TBE electrophoresis through 2% agarose gels and electro-blotting to a nylon membrane. C = No RNA PCR control.

458 I Dayton et al.

the mRNA.12 Prior to this proof, however, there was no assurance that these sequences would be identical. That they were highly homologous, if not identical, was clear since previous Northern blot analyses for B cell IFN-y, when performed under stringency conditions requiring 3 90% identity for hybridization16 showed only a single band of approximately the correct size. Using an equivalent cDNA probe for IFN-y under more stringent conditions, Gray & Goeddel 1~7 found only a single band on Southern blots of human chromosomal DNA cut with the restriction enzyme BamH I. Nevertheless, the possibility of alternative gene splicing could not be ruled out on the basis of these stringency data. Moreover, novel forms of IL-l and TNF-l3 have been recently isolated from B cells.ia~ii As a final consideration, the recently discovered cytokine IL-10 is strikingly homologous to the EBV open reading frame BCRF1.15,is Expression of IL-10 in our B cell lines is almost exclusively restricted to EBV+ lines.15 All of these findings prompted us to sequence the IFN-?I amplicons from three representative cell lines: an EBVf AABCL, an EBV+ non-AABCL and an EBV- undifferentiated lymphoma. If HIV-l can modulate the structure of IFN-y, the AABCL products could conceivably have a different sequence from that of JLP(C). If EBV induces B cells to secrete a cytokine related to but not identical to IFN-y, the IFN-y expressed by the MC116 cell line would be predicted to be different from that of JLP(C). Identifying the identical sequence over the entire protein coding region of the mRNA from T cell clones, NK cells, EBV- and EBV+ B cell lines, confirms that the different lineages express the same IFN-y gene. The original sources of the cell lines did not affect IFN-y mRNA expression. In particular, EBV does not play a role in modulating the expression or structure of IFN-7, a capability EBV may display with other lymphokines.ia,i5,is The production of IFN-), by our B cell lines, as shown by the expression data of this report and the ELISA and RIA data of previous work,879 combined with our recent demonstration that these B cell lines express IFN-y receptors on their surfaces,19 provides support for the existence of autocrine loops as well as paracrine effects for this cytokine. Thus, the influence of IFN-)I on the regulation of the expression of several other cytokines including IL-l,*0 IL-2,21 IL-4,** and TNF-a*s, can be attributed to B cells as well as activated T cells and NK cells. Pleiotropic effects are induced, damped, and augmented by cytokine cascades involved in complex regulatory pathways.24 The extreme complexity of immune regulation by cytokines is becoming more evident as the variety of homologous ligands, multiple receptor subtypes, soluble receptor binding domains and other inhibitors

CYTOKINE,

Vol. 4, No. 6 (November 1992: 454460)

are elucidated. The sum of these studies provides indirect but compelling evidence that B-cell derived IFN-1, may be an important part of the cytokine network which determines the function of B cells, T cells and macrophages. As B cells have also been demonstrated to produce IL-l ,10,*5 an IL-2-like molecule,14 IL-4,*6 IL-5,*7 IL-6,153 TNF-a,29 TNF-p,ii and IL-lo,‘5 B cells should no longer be relegated to the position of passive recipients in lymphokine cascades.

MATERIALS AND METHODS Cell Lines The study included tumor cell lines derived from patients with undifferentiated lymphoma of Burkitt’s and non-Burkitt’s types (N = 21) and normal lymphoblastoid cell lines (N = 7) (Table 1). PA682BM-1, PA682BM2, PA682PE-1, PA682PE-2, PA682PB and AS283 are EBVfMIV-lB cell lines derived from patients with AIDS and American Burkitt’s lymphoma (AABCL). KK124, DW6 and JLP(C) are EBV+ B cell lines derived from patients with American Burkitt’s lymphoma, and EW36, JD38, JD39, CA46, ST486, DS179 and MC116 are EBV- cell lines obtained from American patients with undifferentiated lymphoma of Burkitt’s and non-Burkitt’s types. Raji, Daudi, HRI + 2, Namalva and AK778 are EBV+ cell lines derived from patients with African Burkitt’s lymphoma. All tumor cell lines contain an 8;14 or 8;22 chromosomal translocation. Of the seven normal lymphoblastoid cell lines, three EBVf cell lines were obtained from peripheral lymphocytes of patients with infectious mononucleosis (IM), and three EBV-transformed cord blood (CB) lymphocyte cell lines were derived as previously described.30 PACB-PE is an EBV-transformed B cell line obtained by transfection of normal B cells with supernatant derived from the AABCL PA682PE-1. The cell lines were maintained in suspension culture in RPMI 1640 plus 10% FCS at 37°C in 5% CO,, and were subcultured every 3-4 days. The cell lines have been previously studied for lymphokine secretion and receptor expression,s,14,15,25,26,31-35 and were screened by a panel of B and T cell surface markers. All cell lines were positive for B lymphocyte markers only.

Isolation of T Cells and Neutrophils CD8+ cells from 120 ml whole blood were isolated using CD8 Dynabeads (Robbins Scientific) as described by the manufacturer after initial separation by Ficoll-Hypaque centrifugation and a phosphate-buffered saline wash. The resulting cells were 98% CDS+ by FACS. Cells were primed during the first 24 hours by incubation in OKT3 coated flasks. The medium was RPM1 1640 supplemented with 10% pooled human serum and included 1000 units IL-2/ml. Cells were then transferred to uncoated flasks with continued incubation in IL-2 for 8 additional days. The cells

B cell interferon gamma expression/ 459 displayed good cytotoxic T lymphocyte (CTL) activity at the time of RNA harvesting. Neutrophils from 120 ml whole blood were isolated using a Histopaque gradient centrifugation step. Following the centrifugation, contaminating RBC were lysed with 3% dextran sulfate in 0.9% saline. The remaining neutrophils were pelleted, washed, and then resuspended in the guanidinium denaturing solution for RNA extraction.

Exposure of Cells to Tumor Promoters Cells obtained on the 4th day following subculture were resuspended in fresh medium at 5 X 105 cells/ml and incubated in 75 cm* tissue culture flasks (Corning, Park Ridge, IL) at 37°C for 14 days in the presence or absence of the tumor promoters teleocidin (kindly provided by Dr Fujiki, National Cancer Institute, Tokyo, Japan) or PMA (Sigma, St. Louis, MO). Teleocidin is known to share similar biological activities with PMA and to bind to the same specific cellular receptors as PMA, although structurally it is different.13 PMA may mimic cytokine activity in biological assays. In contrast, no such interference was observed with teleocidin.14 A broad range of concentrations of teleocidin (0.625-10 @ml) and PMA (5-100 rig/ml) was screened previously for effects on cytokine expression. Optimal activation of these B cell lines was obtained with 10 ng teleocidin/ml and with 50 ng PMA/ml, and these concentrations have been used in subsequent experiments. Cultures (control, teleocidin-activated and PMA-activated) were harvested and the cells were immediately processed for RNA studies.

RNA Extraction and RT-PCR Amplification Total cellular RNA was extracted from control, teleocidin-activated, and PMA-activated cells using the guanidinium isothiocyanatelphenol chloroform methodology as previously described.36 We used the RT-PCR technique to detect the expression of IFN-y in B cell lines. Using a Perkin Elmer thermal cycler and the GeneAmp Perkin Elmer PCR Kit (Perkin Elmer, Norwalk, CT) cDNAs were prepared by reverse transcription of 1 l.tg of total RNA using random hexamer primers following the manufacturer’s directions. Then 5-20 1.11of the cDNA reaction was amplified with 2.5 units of Thermus aquaticus DNA polymerase (Tag DNA polymerase, Perkin Elmer) and 20 picomoles each of the 5’ and 3’ primers, in a total volume of 100 1.11.The mixture was overlaid with mineral oil and then amplified over 35 cycles. Each cycle consisted of denaturing at 94°C for 1 minute, primer annealing at 55°C for 1 minute, and extending at 72°C for 1 minute. These primers also contained Hind III (sense) or BamH I (antisense) restriction enzyme recognition sites at their 5’ ends. Primer 1 was comprised of the protein coding nucleotides l-40 (sense strand, 5’-AA AAG C-IT ATG AAA TAT ACA AGT TAT ATC ‘ITG GCT TIT CAG CTC TG-3’) and primer 2 complemented nucleotides 474-501 from the 3’ end of the protein coding region (antisense strand, 5’-GG GGA TCC ‘ITA CTG GGA TGC TCT TCG ACC TIG AAA3’). Amplified fragments were then detected by ethidium bromide staining

after Tris-Acetate-EDTA (TAE) electrophoresis through 2% agarose gels. In some experiments, the amplicons were labeled with [osaP]-dCTP (3000 Ci/mmol, NEN). For these cases, 0.25 1.11[&PI-dCTP per 100 ~1 reaction were added to the standard PCR reaction mix. Following TrisBorate-EDTA (TBE) electrophoresis through 1% agarose gels, the amplicons were transferred by electro-blotting to nylon membranes (GeneScreen Plus, NEN) using a Bio-Rad Semi-Dry Transblot apparatus as described by Lane et a1.37

Cloning and SequencingPCR Products RT-PCR products from amplifications using primers 1 and 2 were directionally cloned16 using T4 DNA ligase (Gibco BRL, Gaithersburg, MD) into the Hind III and BamH I sites of pBluescript II (Stratagene, La Jolla,CA) after passage of the RT-PCR reaction mixture over a NucTrap push column (Stratagene) to remove the unincorporated nucleotides. White colonies growing on LB agar containing 100 pg ampicillin/ml, 40 pg isopropylthio+D-galactoside/ml (IPTG, BRL) and 40 pg 5-bromo-4-chloro3-indoyl+D-galactoside/ml (X-GAL, BRL) were selected and grown overnight in 5 ml of terrific broths8 containing 100 pg ampicillin/ml. Plasmid minipreparations were done following boiling lysisssof the bacteria from 1.5 ml of the overnight culture following the protocol for Strataclean resin (Stratagene) minipreps as described by the manufacturer. Plasmids containing the appropriate size inserts, as determined by electrophoresis in 2% agarose minigels after cutting with Pvu II, were completely sequenced. The SK and KS primers for pBluescript II were used to obtain sequence information for both strands of the insert from double stranded plasmid DNA as described in the Stratagene DSK 35s sequencing kit. Dideoxynucleotide chain terminated products were separated on 6% acrylamide wedge sequencing gels containing 7M urea. Acknowledgements

The authors wish to thank MS Lisa Greenleaf, Dr Jared Klein, Dr Veela Mehta, and Dr Belinda Avalos for generously provided samples of CD8+ T cell and neutrophil RNA and for helpful discussions. We wish to thank Mr Michael Jennings for illustrations and MS Kim Crist for excellent assistance in preparing the manuscript. This work was partially supported by a Davis Fund Scholarship from The Ohio State University Hospitals (MAD), by new program development funds administered by the College of Medicine of The Ohio State University (MAD), by a United States Public Health Service Grant RO-1 AI31262 (DB), and by a grant from the Eastern Star, The Grand Chapter of Ohio (DB). REFERENCES 1. Young HA, Hardy KJ (1990) Interferon-~: Producer cells,activationstimuli, and molecular geneticregulation. Pharmac Ther 45:137-151.

460 I Dayton et al. 2. Pestka S, Langer JA, Zoon KC, Samuel CE (1987) Interferons and their actions. Annu Rev Biochem 56:727-777. 3. Nennesmo I, Olsson T, Ljungdahl A, Kristensson K, Van der Meide PH (1989) Interferon-gamma-like immunoreactivity and T-cell marker expression in rat skeletal muscle fibres. Brain Res 504:30~310. 4. Olsson T, Kristensson K, Ljungdahl A, Maehlen J, Holmdahl R, Klareskog L (1989) Gamma-interferon-like immunoreactivity in axotomized rat motor neurons. J Neurosci 9:3870-3875. 5. Ljungdahl A, Olsson T, Van der Meide PH, Holmdahl R, Klareskog L, Hiijeberg B (1989) Interferon-gamma-like immunoreactivity in certain neurons of the central and peripheral nervous system. J Neurosci Res 24:451-456. 6. Naumovski L, Utz PJ, Bergstrom SK, Morgan R, Molina A, Toole JJ, Glader BE, McFall P, Weiss LM, Wamke R, Smith SD (1989) SUP-HDl: A new Hodgkin’s Disease-derived cell line with lymphoid features produces interferon-y. Blood 74:2733-2742. 7. Taylor MW, Long T, Martinez-Valdez H, Downing J, Zeige G (1984) Induction of y-interferon activity by elevated temperatures in human B-lymphoblastoid cell lines. Proc Nat1 Acad Sci USA 81:4033-4036. 8. Benjamin D, Hartmann DP, Bazar LS, Jacobson RJ (1986) Burkitt’s cells can be triggered by teleocidin to secrete interferon-y. Am J Hematol22:169-177. 9. Pang Y, Norihisa Y, Benjamin D, Kantor RRS, Young HA (1992) Interferon-y gene expression in human B-cell lines: Induction by interleukin 2, protein kinase C activators, and possible effect of hypomethylation on gene regulation. Blood 80:724-732. 10. Rimsky L, Wakasugi H, Ferrara P, Robin P, Capdevielle J, Tursz T, Fradelizi D, Bertoglio J (1986) Purification to homogeneity and NH,-terminal amino acid sequence of a novel interleukin 1 species derived from a human B cell line. J Immunol136:3304-3310. 11. Benjamin D, Kofler G, Tschachler E (1992) Human B-cell TNF-l3 microheterogeneity. Lymphokine Cytokine Res 11:45-54. 12. Gray PW, Leung DW, Pennica D, Yelverton E, Najarian R, Simonsen CC, Derynck R, Sherwood PJ, Wallace DM, Berger SL, Levinson AD, Goeddel DV (1982) Expression of human immune interferon cDNA in E. coli and monkey cells. Nature 295:503-508. 13. Umezawa K, Weinstein IB, Horowitz A, Fujiki H, Matsushima T, Sugimura T (1981) Similarity of teleocidin B and phorbol ester tumour promoters in effects on membrane receptors. Nature 290:411-413. 14. Benjamin D, Hartmann DP, Bazar LS, Jacobson RJ, Gilmore MS (1989) Human B cell lines can be triggered to secrete an interleukin 2-like molecule. Cell Immunol121:30-48. 15. Benjamin D, Knobloch TJ, Dayton MA (1992) Human B cell interleukin 10: B cell lines derived from patients with AIDS and Burkitt’s lymphoma constitutively secrete large quantities of interleukin 10. Blood 80:1289-1298. 16. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual, second edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 17. Gray PW, Goeddel DV (1982) Structure of the human immune interferon gene. Nature 298:859-863. 18. Hsu D-H, de Waal Malefyt R, Fiorentino DF, Dang M-N, Vieira P, de Vries J, Spits H, Mosmann TR, Moore KW (1990) Expression of interleukin-10 activity by Epstein-Barr virus protein BCRFl. Science 250:830-832. 19. Benjamin D (1990) Properties of the IFN? receptor expressed on human B cell lines: identification of high- and intermediate-affinity receptors and 5.6 kb and 8.0 kb IFNy receptor mRNAs. Blood 76 (Suppl):201 (abstr). 20. Donnelly RP, Fenton MJ, Finbloom DS, Gerrard TL (1990) Differential regulation of IL-l production in human monocytes by IFN-?/ and IL-4. J Iminunol145:56~575.

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