CELLULARII~II~UNOLOGY41, 1-8 (1978)

Stimulation of Human Peripheral Blood I_ymphocytes by Autologous EBV-Infected B Cells JEAN VIALLAT, 1 ]~RIK SVEDMYR, F.ITAN YEFENOF, GEORGE KLEIN, AND OLA WEILAND 2

Department of Tumor Biology, Karolinska Instituter, 104 01 Stockholm 60, Sweden Received Jamtary 4, 1978

EBV carrying lymphoblastoid ceil lines (LCL) strongly stimulate DNA synthesis in normal autologous peripheral T lymphoeytes in vitro (autologons stimulation--AS), and generate non-specifically eytotoxic T cells. The AS reaction was explored by replacing the stimulating LCLs with recently infected 13 cells from normal individuals. Autologous/3 cells infected one day earlier with EBV induced significant DNA stimulation but generated no killer cells. The ability to generate cytotoxic T cells appeared 6 to 8 days after EBV infection. Cytotoxicity was not E/3V-speeific. The ability to activate the alternate complement pathway appeared at approximately the same time. /3-cells of convalescent infectious mononucleosis (IM) patients could generate autologous cytotoxicity already 1 day after EBV-infection. The difference between the AS reactions induced by normal and convalescent IM donors is discussed. INTRODUCTION Epstein-Barr virus ( E B V ) converts normal human B lymphocytes that have a limited lifespan in vitro in the absence of mitogenic stimulation, into "immortalized" lymphoid cell lines that proliferate indefinitely in the absence of outside stimulation. In contrast, infectious mononucleosis ( I M ) , an EBV-caused disease, is characterized by self limiting lymphoproliferation in vfvo, benign in character. Although a few EBV-converted, nuclear antigen ( E B N A ) positive cells have been found in the peripheral blood of acute IM patients, they appear to be under host control. This control may be exerted through immunological and/or nonimmunological effectors. Among the former , killer T cells with cytotoxic activity specific for EBV-carrying (but not E B V negative) lines, known to appear during the acute phase of I3/I (1), may be significant, at least in terminating the acute disease. Normal peripheral T-lymphocytes respond in vitro to autologous, EBV-carrying lymphoblastoid cell lines with intense D N A synthesis (designated " A S , " for autologous stimulation) and generate cytotoxic T cells (2, 3). In contrast to the killer T cells of the IM patient, these in vitro generated effectors kill non-specifically, both EBV-carrying and E B V negative targets (4). In a previous paper (5) we have shown that neither careful purification of the effector T-cells nor the pre1 Institut J. Paoli--I Calmettes, C.R.A.C.iVI., 13273 Marseille, France. z Roslagstull Hospital of Infectious Diseases, 114 89 Stockholm, Sweden.

0008-8749/78/0411-0001502A30/0 Copyright © 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

2

VIALLAT ET AL.

treatment of the stimulating LCL by heat or protein synthesis inhibitors improves the specificity of this in vitro system. The present paper explores the nature and the specificity patterns of the AS reaction after replacing the established cell lines with recently E]3V-infected ]3-cells, as the stimulator component. M A T E R I A L S AND METHODS

Lymphocytes. l~resh normal blood was obtained from healthy E]3V seropositive individuals in the laboratory. I-Ieparinized blood from IM patients and convalescent IM donors was received from the Roslagstulls Hospital in Stockholm. Lymphocytes were isolated on Ficoll-Isopaque density gradient (6) and were treated with carbonyl iron and magnetism. T-B separation was performed by E rosette buoyant density fractionation. E rosettes were formed by the method of Jondal et al. (7). E rosette positive cells will be referred to as T cells, and E rosette negative cells as ]3 cells. When necessary, further purification of the T cells was performed by 7S EAC rosetting (Jondal, 8) in order to get rid of Fc- and complement receptor-positive cells. Culture medium. R P M I 1640 (Gibco) was supplemented with L glutamlne (4 X 10-~ M), Streptomycin (100 /~g/ml) penicillin (120 U/ml), Hepes buffer (10 ~2 M) and 10% fetal calf serum (FCS). In place of FCS, human serum from a pool of A]3 + donors, autologous plasma and 13SA (10 mg/ml) were also tested in AS assays. Preparation of Stimulator Cells 13 lymphocytes were exposed to a millipore filtered supernatant of an E]3V-producing 1395-8 culture for 1 hr at 37°C (1 ml/1 × 106 cells), washed, and resuspended in culture medium. Infected cells were grown in stationary suspension cultures in R P M I + 20% FCS and subcultured twice a week. Prior to AS assays, stimulating cells were X irradiated with 3000 R from a Cobalt source. Microassays for [~H]thymidine uptake. Cultures were explanted in Falcon 3040 flat bottomed microplates. 2 to 3 x 105 responder cells and 2 to 3 X 104 stimulator cells were mixed in a total volume of 0.2 ml per well, and incubated in a humiffed 5 ~ COd atmosphere at 37°C for 6 days. 1 ~Ci of [3H]thymidine was added to each culture 18 hr before termination. Microcultures were then harvested on a Skatron harvester (Lierbyen, Norway). Results are expressed as mean counts/rain of triplicate cultures.

Macroassays for Production of Killer Cells Culturs were performed for 6 days in standing Falcon flasks (nr. 3013) containing 7 to 8 × 106 responder cells and 7 x 105 to 15 X 106 stimulator cells. 13efore use for cytotoxieity assay the effector cells were tested for viability and, if necessary, were centrifuged on a Ficoll Isopaque gradient to remove dead cells.

51Cr-Release Cytotoxicity Test The assay was performed in a total volume of 0.2 ml of culture medium in V-shaped micro-titer plates (Linbro, New Haven, Conn.), using graded numbers

AUTOLOGOUS

STIMULATION

FRESHLY EBV INFECTED

WITII

CELLS

3

TABLE 1 Autologous Stimulation (AS) with 1-Day EBV Infected B Cells from Normal Donors Stimulators

[-aH-]thymidine uptake (cpm) Stimulators alone

Responders alone

(3 x 109

(3 × 105)

1-day-uninfected B (a) 1-day-infected B (a)

493 -4- 102

1-day-uninfected B (b) 1-day-infected B (b)

295 -4- 66

Autologous stimulation

37,713 ± 5,706 1,494 4- 435

323 4- 115

67,763 4- 7,272 45,142 -4- 2,898 3,416 ± 835

231 ± 139

60,883 4- 520

3 X l0 ~ responder T cells and 3 X 104 X irradiated i-day EBV infected autologous B cells were cocultivated for 6 days and tested for PH]thymidine uptake. Controls are provided by effector cells cocultivated with uninfected autologous B cells. Two distinct experiments (a and b) are presented. of effector cells and 6 X 103 51Cr-labeled target cells. After 6 to 8 hr incubation, 0.1 ml supernatant was collected for counting. Cytotoxicity was expressed as percent lysis according to the following formula : lysis : 100 ( T -- S ) / ( M a x - S) when T : release in test, S : spontaneous release in medium, M a x = m a x i m u m release in soap. The standard error of triplicate determinations of the percent lysis seldom exceeded five units.

Activation of the Alternate Complement Pathway 5 × 105 cells were incubated with 25 #I normal human serum from an E B V negative donor, in the presence of 10 m a r ethyleneglycol tetra acetic acid and 1.5 m M MgCl2 ( N H S / E G T A - m g 2÷) for 30 rain at 37°C. The cells were then centrifuged and 5/~1 supernatant samples were examined in two dimensional crossed immune electrophoresis (Laurell technique) (9), to detect and quantitate the conversion of C3 into its faster-migrating C3b product. Controls untreated with N H S / E G T A - M g 2+ were also included in the assay.

RESULTS Response of T-lymphocytes from normal, E]3V seropositive donors to the exposure of X-irradiated autologous ]3-cells, E]3V infected one day before was tested by [~i-I]thymidlne uptake after 6-days' culture. In control samples, uninfected B-cells were used. Both the infected and the uninfected ]3 cells stimulated thymidlne uptake considerably, although infected ]3 cells stimulated more strongly, as exemplified in Table 1. No cytotoxic effector cells were generated, however, in any of our several (more than 10) 1-day AS experiments. Similar results were obtained with 2-day infected ]3 cells, as shown in Table 2. In spite of the fact that the number

VIALLAT

ET

AL.

TABLE 2 Autologous Stimulation (AS) with 2 Days EBV Infected B Cells from a Normal Donor [SH]thymidine uptake (cpm)

°fo cytotoxicity Targets (6 × 109

Killer/target ratios

80/1 40/1 20/1 10/i Stimulators alone (3 X 104)

3,417 4- 423

Kaplan (+)

t8

11

5

8

Responders alone (3 × 105)

1,001 =t=

36

Raji (-1-)

17

14

10

5

51,844 =t= 7,367

K562 (--)

1.1

10

2

0

Autologous stimulation (AS)

AS generated effector T-cells were tested for PH]thymidine uptake and cytotoxic response against EBV genome positive (+) and negative ( - ) 51Chromiumlabelled targets. The cytotoxicity patterns observed are similar to that obtained with unstimulated effector ceils. of effector cells recovered after 6 days of culture had increased by a factor of 2, there was no cytotoxic response.

Kinetic Studies W e performed kinetic studies, with stimulator B cells infected between one day and several weeks prior to X-irradiation. Five EBV-seropositive donors were tested by repeated bleedings. Each donor was bled on day 0 to provide B-cells for the infection experiments. Subsequently, they were bled repeatedly to set up AS cultures. As a consequence, each AS culture had to be tested separately for cytotoxicity. In spite of the variation between donors and experimental conditions, it was clear that significant and reproducible cytotoxicity was obtained with 13 cells infected 6 to 8 days prior to the AS experiment (as exemplified in Table 3). There was no detectable EBV-specificity in any of the experiments: E B V carrying and E B V negative lines were killed with equal efficiency. To test the possibility that cytotoxicity might be directed against some F C S associated antigen (10) we have also performed the AS reaction in B S A ( 1 0 / ~ g / m l ) , human A B + serum and autologous plasma, instead of FCS, but without any demonstrable difference compared to the parallel F C S containing cultures. TABLE 3 Cytotoxic Response of Effector T Cells Stimulated with Autologous B Ceils, EBV Infected for 4, 7, and 14 Days Effector T cells stimulated with

% cytotoxicity S J V B (-]-)

K a p l a n (-[-)

K562 ( - - )

4-day-lnfected B 7-day-infected B 14-day-infected B

9 42 47

5 35 37

4 19 29

0 3 9

15 48 78

5 28 52

3 10 29

5 -13

12 29 87

14 27 69

9 17 32

8 6 20

K i l l e r / t a r g e t ratio s

30/1

15/1

7/1

3.5/1

30/1

15/1

7/1

3.5/1

30/1

15/1

7/1

3.5/1

Thi~ table is a collection of undeper~dent A S experiments performed w i t h l y m p h o c y t e s f r o m the same E B V sergpositive donor. S J V B is a n autologous E B V converted line. a G r a d e d n u m b e r s of effector T cells were tested on 6 X 10 8 aCr-labeled t a r g e t cells,

AUTOLOGOUS

STIMULATION

WITI-I F R E S H L Y TABLE

EBV INFECTED

CELLS

4

AS-Response and Cytotoxicity Following Stimulation with B-Cells E B V

Infected

i , 5, 7, and 13 Days Prior to AS Experiment Stimulators

[-3H]thymidine uptake index

% cytotoxicity Targets Sl18 (+)

Uninfected B 1-day-infected 5-day-infected 7-day-infected 13-day-infected

1 2.1 5.9 7.6 15.7

B B B B

Effectors alone (epm)

984

Kaplan ( + )

8 19 31 43 63

0 17 29 38 60

11 3 27 37 46

4 5 24 24 38

43/1

22/1

11/1

5/1

7 I0 18 38 65

1 4 4 25 37

0 0 4 23 37

K562 (--) 0 0 2 17 27

11 18 32 53 56

11 17 24 38 52

14 ll 18 34 45

5 5 17 23 34

K i l l e r / t a r g e t ratio~

Aliquots of a heavily E B V infected B cell culture were frozen d o w n at various stages after infection, and were later t h a w e d on the same day and explanted in A S cultures. A S generated effector cells were tested for [~H]thymidine uptake (expressed by stimulating index) and for cytotoxicity against a panel of EBV-genome positive ( + ) and negative (--) targets. T h e same k i l i e r / t a r g e t ratios were used for each target (6 X 10 a target cells).

In view of the fact that the number of EBV-transformed cells that were present in cultures soon after infection may have been subliminal for the generation of cytotoxic effectors, we have repeated the experiments with two modifications in design: (a) the number of 1-day-infected stimulator cells was increased to a stimulator/responder ration of 1/2; and (b) heavily infected t3 cells were tested after exposure to E B V virus from a batch able to induce in 24 hr 20% E13NA positive cells in Ramos line. On days 1, 5, 7, and 13, culture samples (3 to 5 × 106 cells) were washed, resuspended in 1 ml culture medium + 1 0 ~ DMSO and frozen at minus 90°C. Aliquots were later thawed and explanted in AS cultures. In this way, a single [SH]thymidine uptake and a single 51Chromium release assay could be performed with the various stimulators. As exemplified in Table 4, the difference in kinetic 50

40, SJV8 EBV inf. B cells

o

"~ 30. ta

to20. t)

10.

i

~

I 3

Days

i 4

i 5

following

i 6

, 7

8

9

infection

1716. 1. Alternate pathway activation (Cs conversion) by recently EBV in~ected B-Iymphocytes and by established lymphoid cell lines (Raji, SJVB).

6

VIALLAT ET AL. TABLE 5 AS with 1-day EBV Infected B Cells and T Cells from an IM Donor 8 Days After Illness Targets (6 X 10a)

% cytotoxicity AS generated effector T cells

IM 136 (+) Kaplan (+) K 562 (--)

81 . 69

Killer/target ratio

60/1

79 66

69 . 68

30/1

15/1

.

.

In vivo generated effector

T cells (1) 57

---

-33 35

-17 13

56 7/1

--

33/1

11/1

.

Direct eytotoxicity of peripheral 7S EAC rosette negative T cells (1) was significantly lower than AS generated cytotoxicity. patterns fully confirmed the discrepancy between AS-stimulation and the generation of killer ceils, and the absence of E B V specificity of the eytotoxic response, when obtained. Established L C L s differ from normal B lymphocytes in a n u m b e r of ways. T w o conspicuous differences lie in the ability of L C L to activate the alternate complement pathway (11) and their sensitivity to the non-selective c y t o t o x i c i t y ( N S C ) of F c receptor positive natural killer cells in h u m a n blood ( 1 2 ) . W e wished to test whether the appearance of these two properties coincides with the ability of the B cells to generate cytotoxic T cells in the autologous stimulation experiments. B cells were infected with E B V and tested at various times for alternate complement pathway ( A P ) activation and N S C sensitivity. A s shown in Fig. 1, A P - a c t i r a t i n g ability appears approximately one week after infection, similarly to the ability to generate a cytotoxic response after AS. I n contrast, B cells infected from 1 day to 2 months did not exhibit any sensitivity to N S C , while established lines used as controls were fully sensitive. Therefore, N S C sensitivity appears to be due to m e m b r a n e changes that are not related to either A P activating or AS-killer cell generating ability. TABLE 6 AS with 1-day EBV Infected B Cells and T Cells from a Convalescent IM Donor 10 Weeks (a; b) and 16 Weeks (c) After Illness Targets (6 X 109

% cytotoxicity Effectors T cells (10 weeks) (a)

7S EAC ( - ) T cells (10 weeks) (b)

T cells (16 weeks) (c)

Kaplan (+) K 562 ( - )

39 54

30 49

27 36

23 31

41 62

36 50

20 40

17 38

0 14

0 12

0 5

0 7

Killer/target ratio

60/1

30/1

15, l

7/1.

60/1

30/1

15/1

7/l

60/1

30/1

15/1

7/1

Removal of Fc and Ca receptor positive cells (b) after AS did not abrogate the nonspecific cytotoxicity observed 10 weeks after illness (a).

AUTOLOGOUS

STIIVfULATION

WITILI

FRESIIL¥

E]~V INFECTED

CELLS

7

A S Reaction of T Cells from Convalenscent I M Pat,ients to their Own 1-Dc~y-ln: fected B-Cells In contrast to the findings With the normal donors, 1-day-infected B-cells from convalescent IM donors were able to generate a cytotoxic response after AS. Tables 5 and 6 show the cytotoxicity patterns obtained with donors 8 days (Table 5) and 10 weeks (Table 6; effector (a)) after the acute phase of the disease. As shown in Table 5, the in vivo generated, EBV-spcific cytotoxicity of the same donor was also checked during the acute illness, and was lower than the cytotoxicity generated by AS with the convalescent cells. In spite of this prompt response of the IM patients, the cytotoxicity again was not EBV-specific. Removal of complement and Fc receptor positive cells by 7S EAC rosetting after AS culture (Table 6, effector (b)) did not suppress the cytotoxic response against the EBV-genome negative targets, in contrast to the fractionation experiments performed with the acute IM blood (1). Te AS experiment was repeated with the blood of the same donor 16 weeks after the illness (Table 6, effectors (c)). This time, no killer cells were generated with 1-day-infected B cells. Thus, the ability to generate eytotoxic cells in this system is restricted to the acutely infected patients. DISCUSSION

As in a previous paper (5) we set out to test whether in vitro AS can mimick the EBV-specific T cell response in acute IM patients in vivo. In previous tests with established lymphoblastoid cell lines (LCL) this was not possible. Both EBVseropositive and seronegative donors could generate an AS response that could not be inhibited by anti-EBV sera (13). _AS generated cytotoxicity, was not EBVspecific, since EBV carrying and negative targets were killed equally well (4). Removal of complement receptor carrying cells (2, 3) or purification of effector T cells (5) did not increase the EBV-specificity of the response. Pretreatment of the stimulator LCL, designed to inhibit the release of possible nonspeeifically mitogenic factors (14) did not render the reaction more specific (5). In the present study, we have tested freshly infected B-cells, instead of established LCLs. When 1-day-lnfected B cells of normal seropositive donors were used, a positive AS reaction was obtained with stimulation of DNA synthesis, but, surprisingly, without generation of cytotoxicity. Only 6 to 8 days after infection did the ability to generate cytotoxie, T cells appear. Cytotoxicity was not EBV specific, even after depletion of C3 and Fc receptor positive cells. The inability of 1-day EBV infected B cells to stimulate generation of cytotoxic T cells could not be overcome by infection with more concentrated viruses or by increasing the relative proportion of the stimulating cells. Conceivably, the 6 to 8 days period may have been required to allow the expression of EBV-induced antigens involved in AS, following in vitro infection. One must question whether it is purely coincidental that the same delay after EBV infection is required for B cells to acquire the ability to activate the alternate complement pathway. Experiments are in progress to test whether anti C3 antibodies can block proliferation and/or cytotoxicity in the AS system. This hypothesis of a relationship between alternate complement pathway activation and cytotoxic response is not supported, however, by the finding that 1-day-

8

VIALLAT

lIT AL.

infected autologous B cells of convalescent I M donors were able to generate a cytotoxic response by autologous T cells, however. At this time, it is not clear why the responses of normal and I M lymphocytes differ. One possibility is that B cells of the I1V[ donors have already gone "part of the w a y " in their EBV-transformation in vivo. If this is the case, a phenomenon comparable to in vitro A S must occur in vivo in order to limit further E B V transformation 2 months after illness, when specific killer T cells have already disappeared (1). Alternatively, the difference between normal and I~/[ responses may be determined at the effector T cell level. A way to explain this discrepancy is to imply the existence of an E B V transformation limiting function in vivo, as suggested by T h o r l e y Lawson et al. ( 1 5 ) , and which according to our data is mediated by T cells, does not require macrophage processing and is remarkable for its short range memory

(5). ACKNOWLEDGMENTS This research was supported (in part) by Contract NO1-CP-33316 within the Virus Cancer Program of the National Cancer Institute, the Swedish Cancer Society and King Gustaf V Jubilee Fund. J.V. is a recipient of a DGRST fellowship in Tumor Immunology (Paris). We thank Dr. Mike Scheehy for improving the manuscript. REFERENCES 1. Svedmyr, E., and Jondal, M., Proc. Nat. Acad. Sci. USA 72, 1622, 1975. 2. Svedmyr, E., WigzelI, H., and Jondal, M., Scan& f. [mmunol. 3, 499, 1974. 3. Svedmyr, E., Jondal, M., and Leibold, W., Scan& 1. Immunol. 4, 721, 1975. 4. Svedmyr, E., Deinhardt, F., and Klein, G., Int. ]. Cancer 13, 891, 1974. 5. Viallat, J. R., Svedmyr, E., Steinitz, M., and Klein, G., Cell hnmun.ol. 38, 68, 1978. 6. B/Symn, A., Scan& ]. Clin. Lab. Invest. 21, 97 (suppl.), 77, 1968. 7. Jondal, M., Holm, G. and Wigzell, It., ]. Exp. Med. 136, 207, 1972. 8. Jondal, M., Scan& d. Immunol. 3, 739, 1974. 9. Laurell, C. ]3., Anal. Biochem. 1,0, 358, 1965. 10. Zielske, J. V., and Golub, S. I-I., Cancer Research 36, 3842, 1976. 11. Yefenof, E., Klein, G., and Kvarnung, K., Cell. Immunol. 31, 225, 1977. 12. ]3ak~cs, T., Gergely, P., and Klein, E., Cell. Immunol. 32, 317, 1977. 13. Weksler, M. E., I. ImmunoL 16, 310, 1976. 14. t-Iugues-Law, G., de Gast, G. C. and The, T. I-I. Submitted for publ. 15. Thorley-Lawson, D. A., Chess, L., and Strominger, J. L., ]. Exp. Med. 146, 495, 1977.