Eur. J. Immunol. 1987.17: 1807-1814 T cell-derived tumor necrosis factor 1807

Martin Turner, Mareo Londei’ and Marc Feldmann

Human T cells from autoimmune and normal individuals can produce tumor necrosis factor*

Charing Cross Sunley Research Centre, Hammersmith T cell clones derived from patients with autoimmune diseases were found to be

capable of producing tumor necrosis factor (TNF). This was demonstrated by stikulating the clones, in the absence of accessory cells, with antibodies against the %/ T3 complex and with recombinant interleukin 2 (TL2). Analysis of RNA extracted from these clones showed that TNF mRNA was more abundant than lymphotoxin (LT) mRNA. We also found that TNF protein in the supernatants of these clones was generally more abundant than LT as assessed by using the murine L929 cell assay. TNF production was not limited to T cells from autoimmune individuals, since the T cell tumor HUT78 and T cells purified from the peripheral blood of healthy individu- als also made TNF. Unlike the T cell clones, HUT78 produced greater amounts of LT mRNA than TNF mRNA. Induction of TNF mRNA in T cells from healthy individu- als displayed a two-signal requirement (phorbol myristate 13-acetate and phy- tohemagglutinin or OKT3 and phorbol myristate 13-acetate), similar to that described for the induction of the T cell lymphokines IL2 and interferon-gamma (IFN-y). Additionally we found that IL2 alone was sufficient to induce TNF in these cells when they had been precultured with phytohemagglutinin for 7 days to express IL 2 recep- tors. The cloned T cells we have characterized also produce IFN-y which was detected in the supernatants of the clones using a radioimmunoassay. The evidence suggests that T cells can produce TNF and have the potential to deliver by themselves the dual and synergistic signals of TNFLT and IFN-y to target cells, a process which may be of importance in the pathogenesis of human autoimmunity.

1 Introduction

Tumor necrosis factor (TNF) and lymphotoxin (LT) are pro- teins cytotoxic for certain tumor cells but not normal cells [l]. The genes coding for these mediators have been cloned [2, 31, revealing 28% homology at the protein level and 46% homol- ogy at the nucleotide level, suggesting they are closely related members of a gene family.

Because of the nature of the inducers initially used to produce TNF and the capacity of adherent blood leukocytes and of myelomonoctic leukemias to produce TNF, it has been gener- ally assumed that activated macrophages are the major source of TNF, while lymphocytes are the source of the related LT (e.g. P , 31).

Recently, it has been shown that TNF and LT are more than cytotoxins and have a broad spectrum of activities, such as the activation of polymorphs to phagocytose latex beads [4], stimulation of the growth of fibroblasts [5 ] , prostaglandin E2 and collagenase release from dermal fibroblasts and synovial

[I 63231

* This work was supported by the Nuffield Foundation and Wellcome Trust. Currently supported by the Juvenile Diabetes Foundation.

Correspondence: Marc Feldmann, Charing Cross Sunley Research Centre, Lurgan Avenue, London, W68LW, GB

Abbreviations: CD: Cluster determinant FACS: Fluorescence-acti- vated cell sorter GT: Guanidinium isothiocyanate IL: Interleukin TNF: Tumor necrosis factor LT: Lymphotoxin IFN-y: Interferon- gamma PMA: Phorbol12-myristate 13-acetate PHA Phytohernag- glutinin PBMC: Peripheral blood mononuclear cells FCS: Fetal calf serum PBS: Phosphate-buffered saline

cells [6], and bone resorbing activity [7]. These actions overlap with those of interleukin 1 (ILl), and it is of interest in this context that there are recent reports that TNF can induce blood mononuclear cells [8] or endothelial cells [9] to secrete IL 1. Cachectin, the mediator of the lethal effects of endotoxin and of wasting in cancer patients, has been shown [lo] to be identical to TNF. TNFkachectin is a potent inhibitor of adipo- cyte lipoprotein lipase and this may contribute to the hyper- lipidemia and wasting seen in some patients with cancers and parasitic diseases [ 111.

One of the interesting aspects of TNF and LT is their synergy with interferon-gamma (IFN-y) which has been reported in a number of situations. The best documented is the enhance- ment of cytotoxicity against tumor cells [12, 131. More recently, synergy has been noted in the antiviral effects of these molecules [14], and we have shown augmentation of HLA class IT expression in cells, such as islet cells of the pan- creas, where HLA class I1 expression is not induced by IFN-y alone [15].

We have been interested in the possibility that the synergism between TNFLT and IFN-y may be responsible for some of the manifestations of autoimmunity and may provide a mechanism by which noncytotoxic T cells can modulatehduce the tissue damage noted in these diseases. We have found that T lymphocytes cloned from the lymphocytic infiltrate of tissues of autoimmune diseases released TNF into their supernatants, as well as LT and IFN-y. This result led to an investigation into whether these T cells actually made TNF, a “TNF-like” pro- tein, or an inducer of TNF, by stimulating these T cell clones in the absence of antigen-presenting cells and analyzing their mRNA content. Additionally, we found TNF was produced by a T cell tumor line and highly purified T cells from peripheral blood.

0 VCH Verlagsgesellschaft mbH, D-6940 Weinbeim, 1987 0014-2980/87/1212-1807$02.50/0

1808 M. Turner, M. Londei and M. Feldmann Eur. J . Immunol. 1987.17: 1807-1814

2 Materials and methods

2.1 Reagents, cells and antibodies

Peripheral blood mononuclear cells (PBMC) were obtained from plateletpheresis residues on Lymphoprep (Nyegaard, Oslo, Norway) as described [16]. Phorbol 12-myristate 13-ace- tate (PMA) was purchased from Sigma Chemical Co. (Poole, GB) and phytohemagglutinin (PHA) was from Difco Laboratories (Surrey, GB). Purified recombinant human IL 2 (rIL2) was a gift from Sandoz (Vienna, Austria). OKT3 (anti- CD3), OKT4 (anti-CD4) and OKT8 (anti-CD8) hybridomas were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and antibody was purified from cul- ture supernatants. WT31 antibody was a gift of Dr. W. Tax (Nijmegen, Netherlands). This antibody recognizes a framework determinant of the T cell receptor a/p heterodimer and is therefore specific for all T cells bearing this receptor [17]. UCHTl (anti-CD3) antibody was a gift of Dr. P. Bever- ley (London, GB). Leu 11 antibody (anti-CD16) was pur- chased from Becton Dickinson (Mountain View, CA). OKT3 and WT31 moncolonal antibodies covalently bound to Sepharose 4B were prepared by Dr. E. Zanders (Glaxco, Greenford, GB). The HUT78 cell line was provided by Dr. M. Greaves, and L929 cells were from Dr. E. Simpson (Har- row, GB). HL60 cells were obtained from the ATCC. Cell lines were maintained in complete medium (RPMI 1640 sup- plemented with 10% fetal calf serum (FCS), 2 r n ~ L- glutamine, 100 U/ml penicillin and 100 U/ml streptomycin; all purchased from Gibco-BRL, Paisley, Scotland).

2.2 T cell cloning

This is described in detail elsewhere*. Briefly, T lymphocytes infiltrating tissues (the thyroid gland of a patient with Hashimotos thyroiditis and the synovial membrane of a rheumatoid arthritis patient) were released by collagenase digestion and cultured with rIL2 (20 ng/ml) for 1 week, to expand T cells activated in vivo. T cells were cloned by limiting dilution (0.3 cellslwell) in Terasaki plates (20 pl) in the pres- ence of lo4 irradiated autologous PBMC, OKT3 (30 ng/ml) and rIL2 (20 ng/ml). Clones were maintained by weekly stimulation with OKT3 (30 ng/ml) and irradiated (4000 rds) histocompatible PBMC, and twice weekly stimulation with rIL2 (20 ng/ml).

2.3 T cell rosetting and fluorescence analysis

T cell rosetting was performed as described previously [16]. For immunofluorescence staining, 2 x lo5 cells were incubated for 30 min at 4°C with optimal concentrations of antibodies (UCHTl,OKT8,OKT4, Leu 11). The cells were then washed twice using RPMI 1640 supplemented with 5% FCS and incu- bated with goat anti-mouse IgG fluorescein isothiocyanate (Southern Biotechnology, Birmingham, AL) at a working dilution of 1 : 100 for 30 min at 4°C. After two more washes cells were analyzed using a Becton Dickinson FACStar (fluo- rescence-activated cell sorter).

2.4 mRNA assays

Cytoplasmic RNA was extracted from small numbers (2 X lo6) of cloned T cells using a modified detergent lysis method [18].

* Londei, M. et al., manuscript in preparation.

Cells were washed once with ice-cold phosphate-buffered saline, then resuspended in 100 p1 of lysis solution (10 mM Tris-HC1, pH 8.6, 140 mM NaCl, 1 mM EDTA) containing 10 mM vanadyl complexes (Gibco-BRL). Cells were lysed with Nonidet-P40 at a final concentration of 0.5%. Nuclei were removed by centrifugation for 2 min in an Eppendorf mic- rofuge (Eppendorf Geratebau, Hamburg, FRG). The super- natant was extracted once with phenol and once with chloroform to remove contaminating proteins. RNA was pre- cipitated overnight at -20°C after adding 2.5 volumes of 100% ethanol and sodium acetate at a final concentration of 300 mM. Precipitates were pelleted and dissolved in 50 pl HzO, 20 vl 37% formaldehyde, 3 0 ~ 1 2OxSSC (1 X SSC = 150 mM NaCl + 15 mM sodium citrate). RNA was denatured by heating at 65°C for 15 min then applied in twofold dilutions to a nitrocellulose filter on a Schleicher and Schuell (Dassel, FRG) Minifold 2. Filters were baked for 2 h at 80°C and handled as described for Northern blots.

When greater amounts of cells were available RNA was pre- pared by the guanidinium isothiocyanate (GT) method [19] incorporating CsCl gradient ultracentrifugation [20]. Total cel- lular RNA was electrophoresed through l % agarose denatur- ing gels containing 6% formaldehyde and transferred over- night to nitrocellulose [21]. Filters were baked for 2 h at 8O"C, then prehybridized in 50% formamide, 5 x SSC, 7.5 x Denhardt's solution (1 x Denhardt's = 0.05% Ficoll, 0.05% polyvinylpyrollidone, 0.05% bovine serum albumin), 1 mg/ml denatured salmon sperm DNA at 42 "C for 4 h. cDNA inserts were removed from Vectors by the appropriate restric- tion endonucleases and labeled by random oligonucleotide priming [22] using [a3*P]dCTP from Amersham International (Aylesbury, GB). Hybridization using 3 x 106-5 x lo6 cpdml probe was overnight at 42°C in prehybridization solution. Fil- ters were washed twice in 2 X SSC, 0.1% sodium dodecyl sul- fate (SDS) at room temperature, and twice in 0.2 X SSC, 0.1% SDS at 50 "C and exposed to Fuci X-ray film for 1-4 days with intensifying screens at - 70 "C. Probes were removed from blots by washing for 2 h at 68°C in 5 mM Tris-HC1, 200 p~ EDTA, 0.05% sodium pyrophosphate, 1 X Denhardt's solu- tion. That probes were removed was checked by autoradiogra- phy. In certain experiments autoradiograms were scanned using a Joyce Loebl Chromoscan-3. These results were nor- malized to a control mRNA (7B6) whose expression is invariant throughout the cell cycle [23]. Probes for LT (- 950 bp Eco RI fragment) [2[ and TNF (- 800 bp Eco RI fragment) [3] were kindly provided by Dr. M. Shepard (Genentech Inc., San Francisco, CA). The probe for 7B6 was provided by Prof. U. Torelli (University of Modena, Italy).

2.5 Assays for TNF, LT and IFN-y

The L929 cytotoxicity assay for biologically active TNF and LT was performed essentially as described by Ruff and Gifford [24]. T cell supernatants were incubated with murine mono- clonal antibodies reactive with TNF (neutralizing capacity 2700 UIpg) and LT (neutralizing capacity 550 U/pg; provided by Dr. G. Adolf, Boehringer-Ingelheim, Vienna, Austria) for 1 h at room temperature. Samples were then serially diluted in triplicate and 2 x lo4 L929 cells added to each well. After 24 h plates were stained with crystal violet and washed thoroughly. The dye was solubilized by addition of 33% acetic acid and plates were read on a Titertek (Flow Labs., Rockville, MD) MCC/340 ELISA plate reader. The antibodies were not cross-

Eur. J. Immunol. 1987.17: 1807-1814 T cell-derived tumor necrosis factor 1809

reactive, as anti-TNF did not neutralize highly purified LT from the RPMI 1788 cell line and anti-LT did not neutralize recombinant human TNF (data not shown). One unit of TNF activity is defined as the amount of TNF that causes lysis of 50% of the cells. The concentrations of each cytotoxin present were determined by calculation of titers on cells treated with supernatant alone or with supernatant plus monoclonal anti- body. Internal standards of recombinant TNF were routinely used in this assay. IFN-y in culture supernatants was measured by the Sucrosep radioimmunoassay kit (Boots-Celltech Diag- nostics Ltd., Slough, GB).

3 Results

3.1 Lymphokine production by T cell clones

Analysis of supernatants taken on the last day of the feeding cycle for TNF and LT revealed that there was a predominance of TNF (Table 1) relative to LT with one exception, HTCX25- 72. IFN-I, was detected in varying amounts in all of the sam- ples tested (Table 1).

In order to determine that the TNF was produced directly by the T cell clones, clones were taken 7 days after their last exposure to irradiated PBMC, washed and restimulated in the absence of feeders by solid-phase OKT3 or WT31 and soluble rIL2. Total cellular RNA extracted from 14 clones (8 R.A. and 6 H.T. clones) 8 h after stimulation contained readily detectable levels of TNF mRNA (Fig. 1). A LT probe labeled to a similar specific activity did not hybridize as strongly as the TNF probe (Fig. 1) indicating that LT mRNA was not as abundant as TNF mRNA. Comparison of the amounts of TNF

Figure 1. Lymphokine mRNA production by T cell clones. Two x lo6 cloned T cells were stimulated with rIL2 (20 ng/ml) and either 0.1% OKT3 Sepharose (rheumatoid arthritis clones) or 0.1% WT31 Sepha- rose (Hashimoto's thyroiditis clones).RNA was extracted from T cell clones by the Nonidet-P40 lysis method and from other cells by CsCl gradient centrifugation as described in Sect. 2.4. RNA from unstimu- lated cells taken from the interface after spinning E rosettes on Ficoll served as negative control. HL60 cells were stimulated for the indi- cated times with PMA (50 ng/ml). E+ rosette cells were stimulated with PHA (1 kgiml) and PMA (50 ng/ml) for 8 h. RNA was applied to a slot blot manifold in serial dilutions and treated as described in

mRNA can be made (Fig. 2) after correction for variation in RNA blotted by normalization to 7B6. It can be seen that the

Sect. 2.4,

Table 1. Lymphokine production by cloned T cellsa)

Clone TNFU/ LTU/ designation mlb' mlb)

Rheumatoid RACL 4 26 0 arthritis clones RACL 8 21 3-5

RACL10 3-5 0 RACL 12 32 0 RACL 15 0 0 RACL 18 0 0 RACL 25 6 0

Hashirnoto's HTCX25-30 16 6

clones HTCX25-52 21 11 HTCX2.5-62 19 5 HTCX25-72 10 13 HTCX25-78 0 0 HTCX25-83 14 8

thyroiditis HTCX25-49 17 7

IFN-y Ui ml"

867 919 113 303 482

81 36

446 29 4 4

73 44

358

clones produced different amounts of TNF mRNA and with the exception of HTCX25-18 produced less TNF mRNA than the maximally stimulated HL60 cell line.

Analysis of the T cell clones used in this experiment by immunofluorescence showed them to be a homogeneous population of T cells which did not stain with the NK cell marker Leu-11. All of the clones examined had a CD3+, CD4+, CD8- phenotype; FACS analysis of one such clone is shown in Fig. 3, which demonstrates that there were no residual feeder cells.

Northern blot analysis of RNA extracted from clone HTCX25- 15 showed that 18 S RNA species was present (Fig. 4). This is the same size as the TNF mRNA isolated from PMA-stimu- lated HL60 cells.

3.2 Kinetics of TNF mRNA accumulation in freshly isolated T cells

a) Seven days after their last exposure to irradiated PBMC the super-

tion. The data presented are derived from triplicate determina- tions from a single experiment.

b) TNF and LT were assayed in the murine L929 cytotoxicity assay using monoclonal antisera to distinguish between the two proteins. The concentration (Uiml) of each cytotoxin was determined from titer in the presence or absence of the monoclonal antibody.

c) IFN-y was analyzed by a commercial radioimmunoassay.

E rosette positive (E+) cells (> 85% T cells) from the

PMA and PHA for up to 48 h. Total cellular RNA was extracted and run On a hybridized to an l8 in unstimulated cells (Fig. 5 ) - The level of TNF mRNA peaked at approximately 8 h and returned to nearly basal levels by 48 h. At 8 h an additional band was apparent in the 28 S posi-

natants from T eel1 clones were analyzed for lymphokine produc- peripheral blood of a healthy individual were stimulated with

gel. The TNF mRNA which was

1810 M. Turner, M. Londei and M. Feldmann

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Eur. J . Immunol. 1987.17: 1807-1814

5 0 0 0 d 1 4 8 10 14 2 9 4 4 46 6 11 18 3 0 7 2 7 8 C C

I = s RA CLONES

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Figure 2. Relative levels of TNF mRNA production. Blots from Fig. 1 were scanned using a Joyce Loebl Chromoscan-3. Integrals obtained within the linear range of the film were normalized with respect to the control probe 7B6.

tion (Fig. 5). A 28 S band was also present in stimulated HL60 cells (Fig. 4). This 2 8 s species is unlikely to be ribosomal RNA since it was not found in unstimulated cells. Similar observations have been made by other workers [25, 261 and it has been suggested this represents an unprocessed transcript ~ 5 1 .

3.3 Effect of inducing agents on TNF production by Ef cells

Peripheral blood E+ cells were stimulated by a variety of inducers of TNF. Total RNA was extracted after 8 h and TNF

Figure 4. Northern blot analysis of TNF mRNA derived from a T cell clone. RNA was extracted using the GT/CsCl method from 5 x lo6 cells of Hashimoto clone (HTCX25-15) stimulated with OKT3- Sepharose (0.1% and rIL2 (20 ng/ml) for 8 h, and from HL60 cells stimulated with PMA (50 ng/ml) for 6 h. All of the RNA recovered from the clone (lane 1) and 20 pg total RNA from HL60 (lane 2) were run on a 6% formaldehyde gel, transferred overnight to nitrocellulose and hybridized to the TNF cDNA probe. The blot was then exposed to Fuji X-ray film for 48 h at -70°C.

0 4 8 16 24 48

11632351 HOURS

Figure 5. Induction of TNF mRNA in Et cells. E+ cells were cultured at 2 x lo6 cells/ml in 50 ml complete medium with PMA (1 pg/ml) and PMA (50 ng/ml). RNA was extracted with G T at the indicated times post-stimulation and separated on CsCl gradient. Ten pg total RNA was run on a formaldehyde gel, blotted and hybridized with a TNA cDNA probe as described in Sect. 2.4.

mRNA assayed by slot blot. Supernatants were harvested after 24 h and assayed for TNF in the cytotoxicity assay. Fig. 6 shows that TNF mRNA and protein levels correlated well for most inducers; stimulants that induced the greatest amount of TNF mRNA also induced the greatest amount of bioactivity.

FLUORESCENCE INTENSITY FLUORESCENCE INTENSITY piZ53-1

Figure 3. FACS analysis of clone RACL4. T cell clones used in Fig. 1 were analyzed on FACS. The clone is CD8 (OKT8) and CD16 (Leu 11)- negative, positive staining with OKT3 (CD3) and OKT4 (CD4) results in a shift in fluorescence intensity of approximately one order of magnitude. There are no cells negative for CD3 indicating that the sample is a homogeneous population of T cells. Result is representative; 6 clones (1, 4, 8, 14, 44, 46) chosen at random were analyzed.

Eur. J. Immunol. 1987.17: 1807-1814 T cell-derived tumor necrosis factor 1811

PMA + OKT3

IL2 + OKT3

OKT3

PMA

PHA

IL2 p

PMA + OKT3

IL2 + OKT3

OKT3

PMA

PHA

IL2 1 CONTROL CONTROL

Figure 6. Comparison of various inducers of TNF mRNA and protein in E+ cells. Et cells cultured at 2 X lo6 cells/ml in complete medium with the following agents alone or in combination: rIL2 (20 ng/ml), PMA (50 pgiml), PHA (1 pg/ml), OKT3 bound to the plastic wells of the flask. RNA was extracted 8 h post-stimulation using the GT/CsCl method. Equal amounts of RNA were slot blotted and hybridized sequentially with the TNF and 7B6 probes. Autoradiographs were scanned using a Joyce Loeble Chromoscan-3 and results normalized for the 7B6 probe. Supernatants of duplicate cultures were collected 24 h post-stimulation and assayed for TNF using the L929 cell bioassay as described in Sect. 2.5.

However, while PHA (1 pg/ml) induced modest levels of TNF mRNA there was no detectable bioactivity. PMA induced a similar amount of TNF mRNA as PHA as well as detectable bioactivity. In another experiment, PHA and PMA together produced a strong synergy (data not shown). Solid-phase OKT3, which induces T cell proliferation and the production of other lymphokines [27], induced a greater amount of TNF than PMA alone, but PMA considerably augmented the effect of OKT3. E' cells did not respond well to IL2 alone, which would be expected since few T cells isolated from peripheral blood express the IL2 receptor [28, 291. IL2 did not augment production of TNF by solid-phase OKT3, as did PMA. The failure of IL 2 to induce T cells to produce TNF may have been due to lack of expression of the IL2 receptor. To test this possibility we cultured PBMC with PHA for 7 days to induce IL2 receptor expression, and purified the T cells by E roset- ting. Stimulation with IL2 alone was sufficient to induce TNF (Fig. 7). TNF mRNA induction by IL2 alone approached that found in cells maximally stimulated by the combination of OKT3, PMA and IL2. This suggests that IL2 receptor-bear- ing T cells can produce TNF in response to IL 2.

Five to ten percent of the E-rosetted T cell population could be natural killer cells, some of which are capable of producing TNF [30, 311. Experiments with solid-phase OKT3, which would not be expected to stimulate CD3- NK cells [32], sug- gest that most of the TNF in these samples was produced by activated T cells.

3.4 TNF production by a T cell tumor

The HUT78 cell line derived from a patient with Sezary syn- drome has the phenotype of an activated T cell, expressing cell surface IL2 receptors and HLA class I1 antigens [33]. Super- natants from unstimulated HUT 78 cells contained no detect- able TNF or LT (data not shown). After treatment with PMA these cells produced readily detectable levels of both TNF and

T.N.F.

T.N.F

L.T.

786

~~ 1 6 3 3 1 a b c

Figure 7. IL2 induction of TNF. PBMC were cultured with PHA (1 pgtml) for 7 days then E rosetted. Cells (2 X 106/ml) were either unstimulated (A), or induced with IL2 (20 ng/ml) (B), or IL2 (20 n8/ ml) plus OKT3 plus PMA (50 nglml) (C) and RNA was extracted by the GTiCsCl method and run on a formaldehyde gel. The hybridiza- tion results with both TNF cDNA and the control 7B6 cDNA is shown.

0 4 0 16 24 40

11632381 TIME hrs

Figure 8. TNF and LT mRNA produced by the HUT78 T cell line. HUT 78 cells (2 x 106/ml) were stimulated with PMA (50 ng/ml) over a 48-h time course. RNA was extracted using the GTlCsCl method. Twenty-five pg total cellular RNA was size fractionated on a Northern gel and transferred to nictrocellulose, the probed with TNF and LT probes of similar specific activity. Film exposure time was 48 h.

1812 M. Turner, M. Londei and M. Feldmann

100

> t 0 g 80

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4 0

20

0 -

Eur. J . Immunol. 1987.17: 1807-1814

-

-

-

T

SUPERNATANT

-.-SUPERNATANT

A + ANTI T.N.F.

ALONE

-20 1 I I I

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LOG SERIAL DILUTION [ T i z q

Figure 9. PMA-induced cytotoxins from HUT78 are partially neutralized by anti-TNF. Supernatants collected from HUT78,24 h after stimula- tion with PMA (50 ng/ml), were tested for TNF in the murine L929 cells cell assay as described in Sect. 2.5. The figure shows that monoclonal antibody which neutralized recombinant human TNF considerably reduced the cytotoxicity of the supernatant against the L929 cells.

LT mRNA (Fig. 8). In this cell line the mRNA for TNF was not as abundant as that for LT. Supernatants collected after 24 h of PMA treatment were also toxic to L929 cells, and this could be neutralized in part by monoclonal anti-TNF (Fig. 9).

4 Discussion

Human T cell clones derived from the synovial membrane of a patient with RA and from the thyroid gland of a patient with Hashimoto’s thyroiditis produce both TNF and LT (Table 1). These observations may be relevant to the pathogenesis of the diseases which are characterized by local tissue inflammation and tissue damage, the mechanisms of which are still poorly understood. We were surprised to find that in supernatants from these clones the major cytotoxic activity was neutralized by monoclonal antibody against TNF rather than LT (Table 1). To determine that the TNF was produced directly by the T cells, we stimulated the clones, in the absence of any other cells, by OKT3 or WT31 covalently bound to Sepharose and soluble IL2. FACS analysis verified that the only viable cells were T cells (Fig. 3). Analysis of the mRNA content of activated T cells using cDNA probes specific for TNF and LT revealed that all clones produced mRNA for TNF (Figs. 1, 2), of the appropriate size (Fig. 4). The TNF and LT probes used here detect only single-copy genes [2, 31, suggesting that the hybridization results presented here do not represent the detection of closely related “TNF-like” mRNA. Taken together the monoclonal antibody and mRNA data from auto- immune clones indicate that TNF may be produced by at least some T cells. “TNF-like” molecules produced by alloreactive T cell clones [34], and the T cell tumor HUT102 have also been characterized [35], and many also represent authentic TNF. Thus, TNF production appers not to be solely restricted to cells of the monocyte/macrophage series. Recently, Kobay- ashi et al. [36] have identified a T cell hybridoma capable of producing TNF in response to phorbol esters and calcium

ionophore. These workers, however, did not report whether the fusion partner was capable of producing TNF.

The genes for TNF and LT are located on chromosome 6 in man, and map within the MHC locus, close to the HLA-B region [37]. The two genes are very closely linked in the mouse, being only 1.2 kb apart [38]. Until recently, TNF has been considered the product of activated macrophages, and LT the product of activated lymphocytes [2, 31. A rationale for why two such closely related molecules should be produced by different cells of the hemopoietic lineage has not emerged. Reports predating the cloning of the TNF and LT may have failed to identify the full spectrum of cells producing these molecules since TNF and LT have essentially identical biologi- cal properties, an observation which may be partly explained by the fact that they utilize a common receptor [39]. Recently, TNF production has been reported by other (nonmonocyte) cells including mouse L cells resistant to the cytotoxic effects of TNF [40]. NK cells have been reported to make TNF [30, 311 as has the lymphoblastoid cell line RPMI 1788 [26]. Thus, it appears many different cell types may be capable of producing TNF, in analogy to the many cell types which are now known to produce IL 1 [41] including a recent observation that T cells make IL la [42]. A recent report has shown that blood T cells can make TNF [43]; however, the results with purified cells do not exclude the possibility that NK cells were responsible for the TNF observed.

We were interested in establishing the stimuli necessary to induce T cells to produce TNF. We chose to investigate the production of TNF mRNA and protein by T cells purified from the peripheral blood of healthy individuals. These cells represent a heterogeneous population of resting T cells. Opti- mal induction of TNF mRNA and protein displayed a two- signal requirement, such as OKT3 and PMA. Previous studies on the induction of IFN-y and IL2 mRNA and protein from a T cell line [44] or PHA blast cells [45] have also demonstrated

Eur. J. Immunol. 1987.17: 1807-1814

optimal induction by two signals. These signals activate cal- cium flux and protein kinase C simultaneously [44].

The production of TNF by this mixed population of T cells suggests that TNF production by the T cell clones was not an artefact of the cloning and that TNF production may be a normal consequence of T cell activation. When PBMC were cultured for 7 days with PHA, the cells E rosetting with eryth- rocytes, mostly T cells, many of which were IL2 receptor posi- tive, responded to IL2 alone by making TNF mRNA. This suggests that IL2 alone will induce TNF production by IL2 receptor-positive T cells, and agrees with the findings of Ned- win et al. [46] who showed ILZinduced TNF production in PBMC. We also investigated cytotoxin production by the T cell tumor HUT78. This produces both TNF and LT mRNA (Fig. 8) and secretes TNF (Fig. 9). It is thus similar to HUT 102, another T cell tumor producing TNF activity [35].

The T cell clones we have characterized from autoimmune sites produce IFNy as well as TNF and LT (Table 1). These observations may be important to the pathogenesis of autoim- mune diseases because of the synergy between these lym- phokines. Synergy has been described for their cytotoxicity [12, 131, and, more recently, their antiviral effects [14], class I1 induction [15] and IL1 induction [8]. It is tempting to specu- late that the T cells from the autoimmune individuals are cap- able of delivering the dual and synergistic signals of TNF/LT with IFN-y directly to the target cells. These clones thus have the potential to mediate tissue damage, excess class I1 expres- sion and IL 1 synthesis. In turn, excess class I1 expression and IL 1 production may feed back on the T cells activating them and thus setting up a chronic state of mutual intercellular stimulation. For these reasons the production of TNF by the same T cells that produce IFN-y may be an important aspect of the pathogenesis of destructive autoimmune diseases such as rheumatoid arthritis and Hashimoto’s thyroiditis.

We thank Drs. P. Beverley, G. Adolf and W. Tax for monoclonal antibodies, M. Shepard and U. Torelli for cDNA probes, E. Zanders for monoclonals OKT3 and WT31 conjugated to Sepharose, Sandoz, Vienna for recombinant human IL2, P. Wells for typing the manu- script, G. Dawes, G . Buchan and C. Hewitt for helpful comments, and Dr. Contreras and her colleagues at the National Blood Transfusion Centre, Edgware, for plateletpheresis residues.

Received August 1, 1987.

5 References

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Henzel, W. T., Jarret, J. A, , Leung, D. W., Moffat, B., Ng, P., Svedersky, L. P., Palladino, M. A. and Nedwin, G. E., Nature 1984. 312: 721.

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