Cancer Letters, 7 (1979) 15--20 15 © Elsevier/North-Holland Scientific Publishers Ltd

THE EBV-CARRYING, 32M/Iq..LA DEFICIENT BURKITT LYMPHOMA LINE DAUDI IS SENSITIVE TO EBV-SPECIFIC KILLER T-CELLS OF MONONUCLEOSIS PATIENTS

ERIK SVEDMYb~ and GEORGE KLEIN

Department o f Tumor B~ology, Karohnsha Instltutet, S 104 01 Stockholm 60 (Sweden)

OLA WEILAND Roslagstulls Slu,~hus, Valhallavasen 7, S 114 22 Stockholm (Sweden)

(Received 2 October 1978) (Revised version reeexved 20 December 1978) (Accepted 20 December 1978)

SUMMARY

The/~-microglobulin/HLA deficient Burkitt lymphoma line Daudi was tested for sensitivity to EBV-specific cytotoxicity mediated by natural killer (NK)-depleted T-cells from acute mononucleosis patients. While the Daudi line was not as sensitive as the reference EBV-genome-positive target line, it was clearly sensitive in the majority of cases. This would speak against a major role of syngeneic restriction in this system.

INTRODUCTION

Tursz et al. [13] recently claimed that the 32-mlcroglobuhn/HLA deficient Burkitt lymphoma line Daudi [6,16] is insensitive to the EBV-speciflc, T-cell mediated cytc~toxicit3~ that we previously demonstrated with peripheral T- cells isolated from acute mononucleosis patient~ [1,10,11]. Tursz et ai. have interpreted their finding in light of the syngeneic (HLA) restriction phenom- enon [17]. Since Daudi cells lack HLA, they would be insensitive to T-cell killing, according to th/s argument.

Neither the postulated insensitivity of Daudi nor the postulated HLA restriction agrees with our previous experience on this system [5,10]. While Daudi was not one of the most sensitJvelines, it could be killed by IM-derived T-cells. Moreover, we found that IM-derived T-cells can kill ailogeneic EBV- carrying but not EBV-negative lines. Autologous EBV-carrying Burkitt lym- phoma biopsies or mononucleosis lines provided no obvious advantage over allogeneic EBV-carrying lines.

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Since the claim of Tursz et al+ would have ~mportant consequences, if correct, we have reexamined the sensitivity of the Daudi line to IM-derived T~ells. The results are reported in the present communication.

MATERIALS AND METHODS

Lymphocyte donors Lymphocytes were isolated from the peripheral blood of acute infectious

mononucleosis (IM) patients and normal controls. Clinical diagnosis of IM was confirmed by routine heterophile and other tests, a~ well as a complete assessment of EBV-serology, as described elsewhere [ 11 ].

Lymphocyte separatior, and purification Lymphocytes from heparimzed blood were separated on Ficol-lsopaque

gradients [2], washed 3 times and depleted of macrophages by addition of carbonyl iron and subsequent removal by magnet. As in previous reports [10], natural killer (NK) lymphocytes were removed by a rosette technique specific for Fc and C3 receptor-positive cells [4]. In one case, we have used a more complex three s~;ep method, to isolate Fc-receptor negative T-cells, as described elsewhere [1].

Target cells Three cell l~nes were used: the EBV-genome positive reference line Kaplan

[ 3 ] of IM origin, and the EBV-genome negative line K562 [ 9] o£ myelogenous leukem~ta origin, selected as control because of its hitch sensitivity to NK cell activity and the ~2-microglobulin/HLA deficient Burkitt lymphoma line Daudi [6,16]. Cells were grown m medium RPMI 1640 supplemented with 10% fetal calf serum (FCS) and subcultured 2--3 times weekly.

Cytoto~cicity test A standard SlCr release assay utilizing V-shaped microplates (Linbro) was

used [10]. The incubation period was usually somewhaL longer them specified earlier: i.e., 8--14 h instead of 4--8 h. The target cell rmmber was 6 × 103 and the number of effector cells 10 s and 3 × 10S/well. All tests were per- formed in triplicate. Cytotoxicity was expressed as per cent lysis according to the ~ollowing formula: per cent ]ysis = (a - b)/(c - b) X 100, where a = re- lease in test sample; b = spoutaneous release (medium alone) ;and c --- maximal release as determined by lysis in distilled water (16-24 h) or the highest cytotoxicity value m the test (if higher). Spontaneous release rarely exceeded 35%. Tests with values above 50% were not accepted. The standard error of tl;plicate detezmination was usually less than -+ 5% lysis units.

RE~';ULT~ AND DISCUSSION

In all experiments, 3 cell lines were tested: Kaplart, as the prototype of

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an EBV.carrying, IM-T cell killing sensitive line; K562, as the prototype of an EBV-negative line that is insensitive to IM-T cell killing but is highly sensitive to NK eel]Ls; and Daudi as the ~.mknown'. Table 1 summarizes the results.

As shown in Table 1, uafractionated normal or [M lymphocytes were cytotoxic against all 3 celi~ lines, Kaplan, K562 ancL Daudi, as previously. Depletion of EAC/EA receptor positive cells left a T-cell rich fraction that was depleted of most or al~ ~f the NK-ceU activity against K562, as previously. This fraction was still able to kill the EBV carrying Kaplan line, however, provided it was derived from acute IM. There was no ~sidual effect against Kaplan with similarly puril~ed lymphocytes derived fra.~r~, a case of IM late in convalesence, or lymphocytes from an IM-like (~sease syndrome that was not confn'med serolog~cally as IM or ~ymphocytes from a normal con- trol. All l~his is in agreement with previous experience [10,11].

While the Daudi line was not as sensitive as Kapla~.% ~it was cle~ly sen- sitive in the majority of the cases where the Kaplan line (~ut not K562) was killed by the purified lymphocytes. Particularly iml~ortant is the fact that wlnile the killing effect against K562 decreased upon lymphocyte purification, the killing of Daudl did not, but was ,essentially the same with purified and unpurified cells. The sanle was noted with o~her EBV- carrying lines in our previous study [10].

We must conclude, at variance with the conclusions o~ Tursz et al. [13] that the ~:M/HLA deficier, t Daudi cell is sensitive to IM-T cell killing at least in the majority of the eases tested. This finding is well in line with our previous experiments that showed no obvious syngeneic restriction in this system It is conceivable that this ancient and highl[y efficient virus-ho:~t association may have evolved its own protection mechanisms, without requiring any major help from the HLA system. EBV and its antigenically cross reactive relatives exist not only in all human populations but also in the large apes and in most Old World monkeys (but not in New World monkeys). In spite of its high transforming efficiency in vitro and its ability to induce tumors in New World monkeys, the virus induces only a ]benign lymphopro- liferative disease, mononucleosis, or no disease at all, in man. In the causation of Burkitt's lymphoma, the virus appears ~o play an initiating role, but lym- phoma development itself is due to the combined action of EBV and other factors. The common cytogenetic change (8; 14 tr~L.~;location) appears as the mos~ likely crucial factor that brings about the u ltinmte transformation to autonomous neoplasia (for review see [7] ).

The wate~igh~ protc:t ion against the oncogenic effect of EBV m man resembles the association of other, potentially highly oncogenie viruses with their natural host, such as polyoma and mice, or H. sa;.miri and the squirrel monkey [8]. The efficiency on the host response is also reflected by the very strong ~'n vitro stimulation of autologou¢. ,, peripheral lymphocytes by EBV-transformed B,vell lines, generating a powel~al but not EBV-specific cytotoxieity [12].

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In o u r p rev ious s tud ie s o f t he l a t t e r p h e n o m e n o n [14 ,15 ] we have shown t h a t th i s in v i t ro gene ra t ed c y t o t o x i c i t y a lso fails t o o b e y t he ru les of syngeneic r e s t r i c t i on .

ACKNOWLEDGEMENTS

Th i s p ro j ec t has been f u n d e d in p a r t w i t h f ede r a l funds f r o m t h e Depar t - m e n t o f Hea l th , E d u c a t i o n and Welf;~re o n C o n t r a c t NO1 CP 3 3 3 1 6 , the Swed i sh Cancer Soc ie ty and K i n g G u s t a f V J u b i l e e F u n d .

REFERENCES

1 Bakacs, T , Svedmyr, E., Klein, E., Rombo, L and Weiland, D (1978) EBV-related eytotoxicity of Fc receptor negative T lymphocytes separated from the blood 9f infectious mononucleosis patients. Cancer Letters, 4,183--189

2 Boyum, A. (1968) A one-stage procedure for isolation of granulocytes from human blood Scand J Chn Lab Invest, 21, 51

3 Diehl, V, Henle, G. and Henle, W. (1968) Demonstration of a herpes group virus m cultures of peripheral h,ukocytes from patmnts with infectious mononucleosis J Virol, 2 , 6 6 3 - 665

4 Jondal, M.(1976)Spontaneous~ymphocyte-mediatedcytotoxicity(SLMC)invitro. assay for and removal of the effeetor cell In. In Vitro Methods in Cell-Mediated Immunity, pp. 263- 2,66. Editor Bloom and David. Academic Press.

5 Jondal, M, Svedmyr, E., Klein, E. and Smgh, S. (1975) Killer T cells in a Burkltt's lymphoma biopsy Nature, 255, 405--407.

6 Klein, E, Klein, G., Nadkarm, J., Nadkarni, J., Wigzell, H. and Clifford, P (1968) Surface IgM-kappa specifimty on a Burkitt lymphoma cell iv vivo and in derived culture lines. Cancer Res, 23, 1300--1310.

7 Klein, G. (1975) The Epstem-Barr virus and neoplasia. N. Engl. J. Med., 293, 1353- 1357.

8 K/ein, G and Klein, E. (1977) Immune surveillance against virus-induced tumors and non-rejectabfl~ty of spontaneous tumors -- contrasting consequences of host versus tumor evolution. Proc. Natl. Acad. Sci., 74, 2121--2126.

9 Lozzio, C. B. and Lozzio, B. B. (1975) Human ehro,aic myelogenous leukemia cell line with positive ]Philadelphia chromosome. Blood, 45, 321--323

10 Svedmyr, E. and Jondal, M. (1975) Cytotoxic effector cells specific for B cell hnes transformed by El:,stein-Barr virus are present ~,n patients with infectious mono- nucl[eosis. Proc Na,~l. Acad. Sci , 72, 1622--1626.

11 Svedmyr, E., Jondal, M., Henle, W., Weiland, O , Rombo and Klein, G (1978) EBV specific killer T cells and serologic responses after onset of infectmns mono- nucleosis. J. Cfin. Lab. Immunol., in press

12 Svedmyr, E., Jondai, M. and Leibold, W. (1975) Stimulation of normal lymphocytes with autologous lymphoid cell lines: properties of derived killer cells Scand J Immunol , 4,721~734.

:l 3 Tu~,~, T., Fridman, W. H., Senik, A., Tsapis, A. and Fellous, M. (1977) Human wrus-~nfected t~l'gct cells lacking HLA antigens resist speci~,m T-lymphocyte cytoly,,~s. Nature, 269, 806--808.

14 Via]lat, J., Svedmyr, E., Steinitz, M. and Klein, G. (1978) Stimulation of peripheral human ~ymphocy~es by autologous EBV genome-earrying lymphobl~stmd cell lines Cell Immunol., 38, 68--75.

15 Vial]at, J., Sw~¢lmyr, E., Yefenof, E., Klein, G. and Weiland, O. (1978) Stlmulatmn

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of peripheral lymphocytes by autologous EBV infected 3 cells. Cell. Immunol., in press.

16 Zeuthen, J., Fr~edrich, U., Rosen, A. and Klein, E (197'7) Structural abnormalities in chromosome 15 in cell hnes with reduced expression of beta-2 mieroglobJlin. Immunogeneti~s, 4,567--~ 8n

17 Zinl'.ernagel, R. M. and D~h~rty, P. C (.~t974) Restriction of in ,ntro T-cell-mediated cytotoxicity in lymphocytic chorlomen]ngitls within a syngeneic or semiallogeneic system Nature, 248, 701--702.