ImmmmiogY and Cell Biology (1992) 70, 107-110

Brief Communication A role for tumour necrosis factor-a in the human mixed lymphocyte culture reaction S. J. MATTHEWS and J. S. SULLIVAN NSW Red Cross Blood Transfusion Service, Sydney, New South Wales, Australia Summary The possible role and influence of tumour necrosis factor-a (TNF-a) in the human mixed lymphocyte culture (MLC) reaction was investigated. Polyclona! rabbit sera to reconibinant human-TNF-a (rhTNF-a) consistently inhibited tbe MLC, and this could be reversed by simultaneous addition of rhTNF-a to the culture medium. Under optimal cell culture conditions, the exogenous addition of 1000 U/mL of rhTNF-a had a variable but generally small enhancing effect on the MLC reaction. Additional studies have clearly highlighted the importance of MLC assay conditions in influencing the effect of exogenous rbTNF-a. Enhancement of the MLC reaction was only consistently observed under suboptimal cell culture conditions. Wliiie tbe present study clearly supports an important role for TNF in controlling the proliferative response in the human MLC reaction it also highlights the influence of in vitro tissue culture conditions in determining the effect of exogenous TNF-a in this assay.

Introduction Tumour necrosis factor (TNF) is known to play a central role as a mediator of mflammation. Historically T N F has been associated with tumour regression and endotoxic shock. TNF activity is in fact associated with two distinct poypeptides TNF-a and TNF-P, which display limited sequence homoloev at , - J i l l ? 1 the ammo acid level and, trom an evolutionary c • 1 ^ c Lit point or view, these two factors probably arose by gene duplication.^ In humans these tandemly linked genes have been mapped to the major histocompatibility complex (MHC) region of chromosome 6 and are located approximately 200 kilobases (kb) centromeric of class I genes and approximately 350 kb telomeric of the class III genes.^ Although physically linked at the DNA level, these two cytokines display tissue-specific regulation and are apparently produced by distinct cell types."* Functionally, they can bind to the same recep-

tor and influence a number of common immunological processes, including monocyte activation and control of T lymphocyte proliferation.' Among the documented immunoregulatory functions of TNF-a are its ability ^^ enhance both antigen- and mitogen-induced ^ lymphocyte proliferation,^ upregulate the '[,^^ ^ class II antigens and the high rr ^ IT i . : i Ji n attinity lL-2 receptor, and markedly intJuence , ' . r i i ir • J the generation or both prolireratmg and cyto."^ _ , , r , , ^ ^i, ^ ^°''^^. T ^lymphocytes in the human MLC ^^^^ ^'^"" , I" />oth the murine and human mixed lymphocyte culture (MLC) reactions, TNF-a enhanced the proliferative response, • attbough the extent of tbis enhancement, especially in the human MLC, was highly variable. We hypothesized that this variability might in some way be influenced by tbe MHC haplotypes of the responders and/or stimulators. As a prelude to studying the effects of

Correspondence: J. S. Sullivan, NSW Red Cross Blood Transfusion Service, 153 Clarence Street, Sydney, NSW 2000, Australia. Accepted for publication 23 April 1992.

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S.J. Matthetvs and]. S. Sullivan

TNF-a on MLC responses in fully HLA typed responder/stimulator combinations, tbe effects of TNF-^a on the human MLC reaction using random, untyped responders and stimulators have been reinvestigated.

Materials and methods Cells Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood from healthy donors on Lymphoprep gradients. Isolated PBMC were washed twice and finally resuspended in RPMI 1640 supplemented with 10% human A serum, 25 mmol/L HEPES, 2 mmol/L glutamine, 50 U/mL penicillin, 50 ^g/mL streptomycin and 5 U / mL heparin (culture medium). Proliferative assays Culturing of PBMC was performed at 37''C in a humidified 5% C O T environment for 6 days. Both responders and stimulators were used at a final concentration of 0.5 x 10^ cells/mL (unless otherwise stated). All plate assays were carried out in 0.2 mL volumes. Stimulators were irradiated witb 25 Gy using an irradiator (IBL 437C; Oris Industries, France). Cultures were pulsed with 1 ^iCi of tritiated tbymidine per well 6-18 h prior to harvesting onto glass fibre filters. Sample filters were counted using a Beta-plate counter (LKB-Wallac, Sweden). All experiments were performed in triplicate, results normalized (i.e. presented as a percentage of the control MLC response) and presented as means ± s.e.m. Polyclonal antihuman TNF-a and recombinant TNF-a (rhTNF-a) were diluted in culture medium and added at the initiation of cultures at the concentrations indicated. MLC were set up in either 96-well round-bottomed plates or in 96-well flat-bottomed plates.

Australia; glutamine, penicillin and streptomycin from Fiow Laboratories, Australia, and heparin from Fissons, Australia. Ninety-sixwell flat-bottomed and round-bottomed tissue culture plates were obtained from NUNC, Norway and Flow Laboratories, Australia, respectively. Lymphoprep was obtained from Nycomed, Norway, and tritiated thymidiiie (specific activity 5 Ci/mmol) was obtained from Amersham, Australia.

Results Effect of polydonal anti-TNF-a on MLC The effect of a polyclonal rabbit antiserum directed against rhTNF-a was assessed in a dose-dependent assay in 96-well roundbottomed plates. Varying dilutions of antiTNF-a were added to the MLC at time zero, and proliferation was measured at day 6. As can be seen in Fig. 1, inhibition of MLC by anti-TNF-a was concentration-dependent and the specificity of this inhibition was demonstrated by the ability of simultaneously added exogenous rhTNF-a (1000 U/mL) to overcome this inhibition.

Effect of exogenous rhTNF-a on MLC Using 96-well round-bottomed plates, a total of 16 respoiider/stimulator combinations

Dilution ol anii-TNFc

Materials Polyclonal rabbit antihuman TNF-a was obtained from Genzyme, USA, and rhTNF-a was obtained from Genzyme, USA and Amersham, Australia. RPMI 1640 and HEPES were obtained from Cytosystems,

1/200 1/200 1/SOO 1/eOOO _ 1000 U/mL

Fig. 1. Etfect ot polyclonal anti-TNF-tx on the MLC reaction. These assays were pertonned in 96-weU round-bottomed plates. Results for three MLC combinations are presented as means ± s.e.m.

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Role for TNF-a in the MLC were treated with 1000 U/mL of rhTNF-a with a resultant small increase in proliferation (103 ± 12% compared with the control MLC response). This concentration of rbTNF-a actually inhibited the MLC response in nine of these 16 responder/stimulator combinations. Again using 96-well round-bottomed plates, the effect of varying concentrations of rbTNF-a on the MLC was assessed in an additional 10 responder/stimulator combinations. Tbe MLC responses of 105 ± 6%, 95 ± 6% and 91 ± 6% were obtained in the presence of 100 U/mL, 1000 U/mL and 10 000 U/mL, respectively. Thus over a wide range of rhTNF-a concentrations we were unable to demonstrate an enhancing effect of exogenously added rhTNF-a on the human MLC. Subsequent MLC assays were performed suboptimally by decreasing the numbers of stimulators and/or carrying out the MLC assay in flat-bottomed plates. The MLC responses of 4 separate responder/ stimulator combinations are shown in Table 1. Exogenously added rhTNF-a (1000 U/mL) consistently enhanced the MLC response only under suboptimal culture conditions (Table 1). It is worth noting that MLC responses in round-bottomed plates were higher than flat-bottomed plates and were also higher than the enhanced rbTNF-a-mediated MLC responses in flat-bottomed plates in three of the four responder/stimulator combinations (Table 1). This would indicate that culture conditions in the round-bottomed plates were optimal and that under sucb conditions rbTNF-a does not enhance the MLC response.

Discussion TNF-a is a pleotrophic cytokine that play.s an important role in modulating the cellular immune response.' More recently it has been shown to influence proliferative responses in both the murine and human MLC reaction. • In the present study, the role for TNF-a in the human MLC assay has been reinvestigated. Previous work has demonstrated an important role for TNF-a in modulating tbe proliferative response of allogenically stimulated PBMC/' and while our work also supports this idea, we were initially unable to demonstrate a consistent enhancing effect for exogenously added rbTNF-a In MLC. Despite this the essential role for TNF-a in the human MLC was confirmed,^ as anti-TNF-a inhibited the MLC and rhTNF-a reversed this inhibition. Our initial failure to reproduce rhTNF-a-induced enhancement ot the MLC^ was essentially due to sligiit differences in experimental conditions employed in the two studies. Suboptimization of MLC assay conditions decreased tbe proliferative responses compared with those obtained under optimal conditions and more importantly rendered these cells responsive to tbe enhancing effects of exogenously added rhTNF-a. A related study of tetanus toxoid presentation by fixed macrophages to purified CD4 * lymphocytes demonstrated that rhTNF-a mediatedenhancement of T cell proliferation was only observed in flat-bottomed plates (i.e. suboptimal assay conditions).'' It appears that round-bottomed plates maximize cell-to-cell contact, and such contact appears to optimize proliferative responses in the MLC assay. The

Table 1. Comparison of effects of TNF-a on the MLC reaction in round-bottomed and flat-bottomed 96-well plates Flat-bottomed wells Round-bottomed wells (5x 10' Stimulators/mL) (5 X 10^ Stimulators/mL) (5 X 10" stimulators/mL) MLC ]MLC + T N F ' MLC + TNF-a C)^i.) MLC MLC + T N F ' MLC + TNF-a (%) MLC MLC + T N F ' MLC + TNF-a (%) (cE/minxiO ') MLC ( a / m i n x l O •*) MLC: (ct/min X 10 ^) MLC 82.0 70.2 87.2 99.9

70.0 71.2 63.5 98.8

• 1000 U/mL rhTNF-a.

85 101 73 99

50.4 30.9 34.2 79.9

68.9 40.1 53.8 104.5

137 130 157 131

3.5 1.0 1.7 6.0

7.2 3.0 3.2 6.1

206 300 188 102

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S.f. Matthews and]. S. Sullivan

importance of fully describing cell culture conditions in studies of biological response modifiers (such as cytokines) cannot be overemphasized, and this study serves as a reminder that sligbt variations in experimental conditions can bave a profound influence on the actual effects observed with recombinant cytokines on in vitro cultured cells. Furthermore we would suggest that in vitro studies of biological response modifiers should routinely be carried out under botb optimal and suboptimal cell culture conditions.

References

1. Beutler, B. and Cerami, A. 1989. Ami. Rev. Immunol. 7: 625-655. 2. Nedospasov, S. A.. Shakhov, A. N., Turetskaya, R. L. e{ al. 1986. Cold Spring Harbor Symp. Quant. Biol. 51: 6\ 1-62?,. 3. Carroll. M. C , Katzaman. P., Alicot, E. M., et al. 1987. Proc. Natl Acad. Sci. USA 84: 8535-8539. 4. Anderson, U., Adolf, G.. Dohlsten, M. et at. 1987./ Immunol. Methods 123: 233-240. 5. Yokoto, S., Geppert, T. D. and Lipsky, P. E. 1988./ Immunol. 140: 531-536. 6. Scheurich, P., Thoma, B.. Ucer. U. er al. 1987./ Immunol. 138: 1786-1790. Acknowledgements 7. Shalaby, M. R., E^pevik, T., Rice. G. C. et al. 1988./. Immutiol. 141: 499-503. We are grateful to J. Trejaut, H. Dunckley, C. 8. Robinct, E., Branellec, D., Termijtelen, A. M. el Farrell and A. Geczy for their helpful comal. 1990./ Immunol. 144: 4555-4561. ments and assistance. This work was sup- 9. Talmadge, J. H., Phillips, H., Schneider, M. et al. ported in part by a grant from the NH&MRC. 1988. Cancer Res. 48: 544-550.

A role for tumour necrosis factor-alpha in the human mixed lymphocyte culture reaction.

The possible role and influence of tumour necrosis factor-alpha (TNF-alpha) in the human mixed lymphocyte culture (MLC) reaction was investigated. Pol...
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