Studies on the mechanism of measles virus-induced suppression of lymphocyte functions in vitro

CELLULAR

37, 44&458 (1978)

TMMUN~L~OY

Studies on the Mechanism

of Measles

of Lymphocyte

Virus-Induced

Functions

Lack of a Role for Interferon

Suppression

in Vitro

and Monocytes

l

C. J. LUCAS, Josk UBELS-POSTMA, J. M. D. GALAMA, AND ANNA REZEE Celttral Laboratory Laboratory

of the Netherlands Red Cross Blood Transfusion Service and the of the University of Clinical and Experimental Immunology of Amsterdam, Amsterdam, The Netherlands Received January 5,197s

In a previous paper it was reported that an inhibitory effect of measles virus on lymphocyte transformation is only observed if replication of measles virus can take place. We subsequently investigated whether virus-induced interferon could be held responsible for the suppressed lymphocyte transformation. This led to the conclusion that virus-induced extracellular interferon is probably not responsible for the observed suppression, since uv-irradiated or ultracentrifuged supernatants of infected lymphocytes did not inhibit. Furthermore, inhibition of the mixed lymphocyte reaction (MLR) occurred even in the presence of noninfected monocytes. This makes it unlikely that monocytes are responsible for mediating the virus-induced immunosuppression. In this study we also investigated whether a lymphocyte function, which does not require cell multiplication, is inhibited by measles virus. The stimulating capacity of lymphocytes infected with measles virus in the MLR was found to be significantly diminished under conditions that exclude infection of the responding cells.

INTRODUCTION Previously, we demonstrated that live measles virus inhibits DNA synthesis and blastogenesis of stimulated lymphocytes (1). However, the mechanism leading to this suppression is still far from clear. Several other viruses also inhibit lymphocyte stimulation, e.g., polio virus, herpes simplex virus, and rubella virus. The inhibition by polio virus and herpes simplex virus is due to the infection of monocytes by these viruses (2, 3). Several reports have indicated that monocytes play an essential role in lymphocyte stimulation in vitro (4, 5), although their exact function is not yet understood. Virelizier suggested, on the basis of these observations, that many virus-induced immunosuppressive effects are mediated through the interference with monocyte functions (6). The experiments described in this article, however, will demonstrate that, in the case of suppression by measles virus, infection of monocytes does not play an important role. IThese studies were supported by a grant from FUNGO, Grant No. 13-38-13. 448 OOOS-8749/78/0372-0448$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

The Hague, The Netherlands,

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449

It has now become clear that interferon can have a regulatory function in the immune response (7, 8). It is possible that the observed inhibition is due to virusinduced interferon rather than to the measles virus itself. However, in the present study, it could be excluded that extracellular interferon, produced by infected lymphocytes, is responsible for the observed suppression. It is not known whether measles virus has an inhibiting effect only on the stimulation of lymphocytes as measured by thymidine incorporation or also on other lymphocyte functions. Therefore, the effect of measles virus on the capacity of infected lymphocytes to stimulate allogeneic lymphocytes in the mixed lymphocyte reaction was investigated. It appeared that the stimulatory capacity of lymphocytes infected with measles virus was impaired. We also investigated the effects of measles virus on cytotoxic effector functions of lymphocytes. The results of these experiments will be published separately. MATERIALS

AND

METHODS

Virus Measles virus Edmonston B strain was obtained from Dr. R. Brouwer, Institute for Public Health, Bilthoven, The Netherlands. The virus was grown in VERO cell monolayer cultures, which were free of mycoplasma. VERO cells were grown in medium 199 with Earle’s salt solution containing 20 mM bicarbonate and supplemented with 10% fetal bovine serum (FBS) .2 Penicillin (100 U/ml) and streptomycin (100 pg/ml) were present and used throughout. Virus was harvested by collecting supernatant media from infected cell cultures in roller bottles, which were infected at a multiplicity of infection (m.o.i.) of 0.001. The supernatants were centrifuged for 10 min at 1600 rpm to remove remaining cells. Virus stocks were kept at - 70°C. Virus titers ranged from lo6 to lo7 tissue culture infection dose ( TCID50) /ml. V’ lrus titrations were performed on VERO cell monolayers in microtest tissue culture plates with flat bottoms (Greiner, S. A.). Confluent monolayers were infected eightfold with 200 ~1 from twofold dilutions of measles virus and tested for cytopathological effects. Plates were incubated in a humidified atmosphere containing 5% CO2 at 37°C. The titer, i.e., the dilution at which 50% of a series of cultures was infected, was calculated according to the method of Reed and Muench. Inactivation was performed either by heating the virus suspensions at 56°C for 1 hr, or by treating it with ultraviolet irradiation (35,000 erg/mm?). Before being added to lymphocyte cultures, measles virus was diluted with RPMI-1640 medium containing 25 mM Hepes (Gibco) . Lymphocyte Separation,

Culture, and Infection

Peripheral blood lymphocytes were obtained from defibrinated blood by centrifugation on a Ficoll-Isopaque layer with a density of 1.079 g/ml at 22°C. The collected lymphocytes, which still contained 10-30s monocytes, were washed twice with medium containing 5% FBS. All blood donors were healthy adult volunteers who were seropositive to measles virus as tested by virus neutralization or com* Abbreviations used: PHA, phytohemagglutinin; MLR, mixed lymphocyte reaction ; TCID,, tissue culture infective dose; FBS, fetal bovine serum; Hepes, 4- (Z-hydroxyethyl) -l-piperazineethane sulfonic acid; TdR, thymidine ; FDA, fluorescein diacetate; m.o.i., multiplicity of infection ; Con A, concanavalin A.

450

LUCAS

ET

AL.

plement fixation. In some experiments, the blood was treated with carbonyl-iron particles (2 mg of carbonyl-iron/ml of blood) to decrease the monocyte content of the resulting lymphocyte suspension. After this procedure, the lymphocyte suspension contained only 2-5 % monocytes. The amount of virus added per lymphocyte is given as multiplicity of infection which means the number of infectious virus particles added per lymphocyte. After infection the virus was left with the lymphocytes, unless otherwise indicated. Lymphocytes ‘were cultured in round-bottom microtest plates. In each well, 4. lo4 lymphocytes were incubated in 150 ~1 of medium. The medium was RPMI-1640, bicarbonate-buffered and supplemented with 15 % FBS unless otherwise indicated. Penicillin (100 U/ml) and streptomycin (100 rg/ml) were present in all media and solutions. Phytohemagglutinin (PHA, Wellcome) was added to a final concentration of 50 pg/ml. Concanavalin A (Con A) was used in two different concentrations depending on the source of serum in the tissue culture medium. When cultures were performed in FBS, 6 pg of Con A/ml was used as final concentration, whereas in human serum at 60-pg/ml final concentration was optimal. Lymphocytes cultured with PHA or Con A were usually incubated with 3 or 4 days unless otherwise indicated. In one-way mixed lymphocyte reactions, 4. lo4 irradiated stimulator cells (2000 rad) and 4. lo4 responder cells were cultured in 150 ~1. Synthesis of DNA was measured by adding 20 ~1 of [3H] thymidine (200 mCi/mmol, 20 &i/ ml) 24 hr before cultures were harvested. Cells were collected on glass-fiber filters. The washed filters were dried and counted in a liquid scintillation counter, in standard scintillation fluid. When large numbers of lymphocytes were required, 3. lo6 cells were cultured in 4 ml or 3. lo5 cells in 1 ml. In these experiments medium RPMI-1640 buffered with 20 mM Hepes was used. Viability Cell viability of nonstimulated cells was measured by trypan blue exclusion. Cell viability of stimulated cells was measured by the uptake and conversion of fluorescein diacetate (FDA) by living cells (1). Cells were incubated with FDA (25 pg/ml) for 5 min. Under a fluorescence microscope, 200 cells were examined to permit discrimination between stained and nonstained cells. Propidium iodide was used as counterstain. Interferon

Titrations

Titrations of interferon in the supernatants of lymphocyte cultures were performed by Dr. A. Billiau, Rega Institute, Leuven, Belgium. Details of the assay have been described elsewhere (9). In short, viruses were inactivated by decreasing the pH of the solution to 2; subsequently, interferon was assayed on Hep-2 cells which were challenged with vesicular stomatitis virus. Cells were grown in flatbottom microtiter plates, and the amount of CPE was judged after the cells had been stained with 0.5% crystal violet. Results are expressed as reference units per milliliter in terms of the NIH reference preparation 69/19. Neutralization

of Measles Virus

Heart-inactivated serum was titrated by the following method for the presence of antibodies with the ability to neutralize measles virus. A measles virus sus-

MEASLES

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LYMPHOCYTE

TABLE PHA Stimulation Lymphocyte donor

A B C D

451

FUNCTION

1

of Lymphocytes

Infected

with

Virus-infected cellsa (cpm X 10ea)

Noninfected cells (cpm X lo-“)

1.6 1.4 4.4 5.3

40.2 12.6 38.9 45.0

Measles Virus Inhibition (70)

96 89 89 88

a Lymphocytes were infected with measles virus 96 hr before PHA was added. Virus was added at m.o.i = 2. The incubation with virus was performed in fetal bovine serum; after addition of PHA, 20% pooled human serum was present.

lo3 TCIDsO was incubated with twofold dilutions of a serum. containing After 1 hr of incubation at room temperature, the samples were kept at 4°C for 16 hr. Samples were tested eightfold for CPE on VERO cell monolayers in microtest wells, and the neutralization titer ( NTsO) was determined.

pension

RESULTS of Measles Virus on the Stiwmla-

1. Role of Monocytes with Regard to the Efect tion of Lymphocytes

As will be shown in a forthcoming paper (lo), it is possible to infect lymphocytes in vitro in the absence of a known stimulus. When such infected lymphocytes are subsequently cultured in the presence of antibodies against measles virus, an almost complete inhibition of PHA-induced lymphocyte transformation was observed (Table 1). TABLE PHA Stimulation

2

of Mixtures of Noninfected Lymphocytes Infected with Measles Virus

Number of cells infected with measles virus (X 10-Y

Number of noninfected cells (X 10-Y

0 10 20 30 40 40

40 40 40 40 40 0

C3H]Thymidine Cultures performed in pooled human serum (cpm X 10p3)

39.3 41.2 41.6 45.56 44.76 0.7

21.0 27.2 29.7 3S.2b 36.9” 0.2

and Lymphocytes

incorporated Cultures performed in fetal bovine serum (cpm X lo-“)

33.3 12.5 15.0 14.3 13.8 2.0

22.6 6.6 7.1 9.1 10.0 0.9

0 A and B indicate lymphocyte suspension from two different blood donors. b Whether the slightly enhanced DNA synthesis of these mixtures is the result of a feeder effect or stimulation of a measles virus-specific lymphocyte is not yet known. We prefer to think in terms of a feeder effect.

452

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ET AL.

TABLE Responding

Capacity

in the MLR

Responding Virus-infected 7,661 f 4,819 f

of Lymphocytes

capacity

cells” 1,243 4.51

3 Infected

(cpm)

Inhibition (%I

Noninfected 25,329 f 13,712 f

with Measles Virus

cells 2,794 2,298

70 65

0 Lymphocytes were infected with measles virus 96 hr before irradiated allogeneic cells were added. Virus was added at m.o.i. = 2. After the incubation with virus, performed in fetal bovine serum, the allogeneic cells were added and the serum replaced by 20% pooled human serum.

In an earlier paper (1) we reported that antibodies against measles virus prevent spreading of infectious measles virus in lymphocyte cultures. Additional proof was obtained in the following experiments. When mixtures of infected and noninfected lymphocytes were cultured with PHA, it was observed that the addition of 40,000 infected lymphocytes had no effect on the stimulation of 40,000 noninfected cells (Table Z), so that secondary infections are very unlikely when cultures are performed in human serum containing antibodies against measles virus. These observations allow us to investigate a possible role of monocytes in the observed inhibition induced by measles virus. Lymphocytes infected with measles virus were stimulated 4 days after infection with irradiated allogeneic cells in the presence of human serum containing antibodies against measles virus. This serum prevents spreading of the infection to the monocytes present in the suspension of stimulator cells. Table 3 shows that, under these conditions the mixed lymphocyte reaction is still inhibited considerably. Similar results were obtained when autologous irradiated, noninfected cells were added to infected, nonstimulated lymphocytes. These cell mixtures were stimulated with concanavalin A, since the activity of this mitogen is strongly dependent on the presence of monocytes. Table 4 shows TABLE Stimulation

by Concanavalin

A of Mixtures

4 of Virus-Infected Inhibition

Cells

(%)

in

Ba, cultured

Pooled human serum

FBS

Pooled human serum

67

87

60

Aa, cultured

Virus-infected lymphocytes only* Virus-infected lymphocytes + irradiated autologous cellsc

And Noninfected

57

in FBS

82

59

a A and B indicate lymphocyte suspensions from two different blood donors. * Lymphocytes were infected with measles virus 96 hr before irradiated cells and Con A were added. c A number of irradiated cells (4000 rad) equal to the number of infected cells was used. Cells were isolated from a fresh sample of blood from the same donor.

MEASLES

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453

that the inhibition by measles virus is slightly, but not significantly, diminished. This means that the presence of irradiated noninfected monocytes does not counteract the inhibitory activity of measles virus. 2. Inhibiting

Efect of Supernatants from Infected Lymphocyte Cultures

Experiments were designed to test whether the suppression of lymphocyte stimulation is caused by measles virus itself or by virus-induced interferon. Supernatants from infected, PHA-stimulated, lymphocyte cultures were added to new cultures of lymphocytes from the same individual to see whether they were inhibitory. Moreover, the concentrations of infectious measles virus and interferon were determined in these supernatants. In Fig. 1 the extent of inhibition of stimulation

oglOvirus titer

%inhibition

100

80

60

40

6

20

I

24 72 96 40 time of supernatant collection in original culture FIG. 1. Relation between measles virus titer in lymphocyte culture supernatants and the extent of inhibition caused by this supernatant in other lymphocyte cultures. Untreated supernatant (25 pl) from PHA-stimulated and infected or noninfected lymphocyte cultures was added together with PHA to new cultures of cells from the same donor. The mean of the results obtained in two different cultures in one experiment is shown. Several other experiments gave similar results. Abscissa, original culture period ; ordinate, left, titer of measles virus expressed as log TCIDm per milliter; ordinate, right, extent of inhibition expressed as

(

counts per minute, culture + measles virus x 100 yo, counts per minute, culture - measles virus > O-O, virus titer; the mean results obtained in two different cultures in one experiment are given. O-O, inhibition of second culture; mean results obtained with superuatants from two different cultures in one experiment. 100 -

454

LUCAS

ET

TABLE Effect

AL.

5

of Measles Virus from Supernatants Lymphocytes on MLC

of Infected, Reactivity

PHA-Stimulated

Time of supernatant collection after PHA addition0 (hr)

MLR

(cpm X 10-r)

A”

Without

supernatant

added

Bb

20.6

22.2 21.8 21.6

24

Noninfectedc Infected Percentage inhibition

14.5 11.6

48

Noninfected Infected Percentage inhibition

12.4 1.9

72

Noninfected Infected Percentage inhibition

14.1 1.5

96

Noninfected Infected Percentage

16.7 2.4

20

22 26.6 3.2

85

88 25.6 3.3

89

inhibition

87 28.8 3.7

86

87

QSupernatants were obtained by centrifugation of the cells and taking 2-3 ml from the medium. Supernatants were stored at -70°C until use. b Supernatant from A lymphocytes stimulated by PHA was added to cultures where A was the responder cell in the unilateral MLR. A and B indicate lymphocyte suspensions from two different blood donors. c To each well of the microtest plates, 25 ~1 of supernatant was added.

by PHA of cells from the same donor by the untreated supernatant of infected cultures is compared with the virus titer. The time curves are similar. The supernatant of infected PHA-stimulated lymphocytes also appeared to inhibit MLR activity (Table 5). The concentration of interferon was measured at two different time intervals after infection and stimulation of the lymphocytes, respectively. After addition of either PHA, measles virus, or both to lymphocyte cultures, quantitatively similar interferon concentrations were found after 24 or 72 hr. The highest concentration TABLE Interferon

Production

Condition

Lymphocytes Lymphocytes Lymphocytes

6

in Lymphocytes Infected and Stimulated with PHA

+ measles virus” + PHA + PHA + measles virusC

with Measles Virus

Interferon

titer in reference unitsa*b 1.7 1.4 2.3

5 Mean results of four determinations obtained in different leukocyte cultures. h Identical results were obtained with supernatants taken 24 or 72 hr after the addition and mitogen. c Measles virus added at m.o.i. = 2.

of virus

MEASLES

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TABLE Properties

Treatment

455

FUNCTION

7

of Inhibiting Factor from the Supernatants PHA-Stimulated Lymphocytes

of Infected,

Inhibition

of supernatant’

(‘%) Unilateral

PHA

None uv Irradiation6 Heat inactivationC Ultracentrifugationd

MLR

Ab

B

Ab

B

83 11 0 4

81 13 8 8

89 18 18 24

87 2 14 21

0 To each well of microtest plates, 25 pl of supernatnat were added together with PHA or stimulator cells. b A and B indicate lymphocyte suspensions from two different donors. Supernatants were obtained from and added to cultures of cells from the same donor. c As described in Material and Methods. d Centrifugation was performed in the SW 60 LTI rotor in a Beckman L2-65B ultracentrifuge. Samples were centrifuged for 10 min at 45,000 rpm.

of interferon was found in cultures of infected, PHA-stimulated lymphocytes (Table 6). To establish whether the inhibition of lymphocyte activity by these supernatants was due either to measles virus or to interferon, we inactivated infectious virus by uv irradiation or removed it by ultracentrifugation. Heat-inactivation, uv irradiation, and ultracentrifugation completely removed the inhibitory activity, although some of the treated supernatants still slightly inhibited the MLR (Table 7). Since interferon should resist uv-irradiation and ultracentrifugation, we conclude that measles virus was indeed responsible for the observed inhibition. 3. Stimulating

Caspacity of Lymphocytes Infected with Measles Virus

The same observations that allowed us to determine the role of monocytes also allowed us to study the effect of measles virus on the capacity of lymphocytes to stimulate allogeneic lymphocytes in the MLR. Lymphocytes which were infected TABLE Stimulating

Versus Responding

Capacity

8

of Lymphocytes

Cell donor

Inhibition PHA stimulation

A B C D n*b Mean results suspensions.

with

two different

44 67 58 42 allogeneic

with Measles Virus

(rO)

Responding capacity MLRQ

84 84 93 91 obtained

Infected

stimulator

Stimulating capacity MLRb 78 70 69 31 (u) or responder

(0) cell

456

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ET AL.

with measles virus during a 96-hr preincubation were irradiated with 2000 rad and then tested for their stimulatory capacity. Each stimulator cell suspension was tested with two different unrelated responding cell suspensions. The results of four experiments in which infected irradiated lymphocytes were compared with noninfected cells are shown in Table 8. The responding and the stimulatory capacities of infected lymphocytes were inhibited to similar extents. DISCUSSION In a previous paper we reported that live measles virus inhibits DNA synthesis in stimulated lymphocytes (1). This is most likely accomplished by virus functions for the expression of which lymphocyte stimulation is needed (1, lo). In this paper we have extended these observations by excluding a role for extracellular interferon as the inhibiting mediator, Moreover, the supposition that the presence of monocytes is a prerequisite for this inhibition turned out to be unlikely. Furthermore, we observed that the stimulatory capacity of lymphocytes in the MLR was considerably inhibited after infection with measles virus. Leukocyte interferon can be induced by both mitogens (11) and viruses (12). In different assays, interferon had a regulatory effect on lymphocyte functions (7, 8, 13). In this study, however, we found no effect of exogenous, virus-induced interferon on the transformation of lymphocytes. Mitogen-induced interferon had no influence either on the stimulation or the proliferation of lymphocytes, the slight inhibition observed in experiment A, Table 1, being an exception. These interferons should be mentioned separately since it has been observed that mitogeninduced interferon differs from virus-induced interferon, even if synthesized by the same cells (12). No clear study as to whether both leukocyte interferons can regulate lymphocyte reactivity is known to us. Our inability to observe an inhibiting effect of interferon in supernatants of lymphocyte cultures is most likely explained by quantitative differences between this study and others. In our hands, these experiments served as controls to the experiments performed with supernatants containing measles virus. It may be that higher concentrations of interferon are inhibiting. The results when taken together mean that the inhibition, obtained in lymphocyte cultures to which minute amounts of supernatants from previous cultures of the same, but measles virus-infected, lymphocytes were added, is fully explained by infection with measles virus. On the other hand, it is still possible that within the infected lymphocyte the inhibition of transformation is mediated by virus-induced intracellular interferon-like substances. Virelizier (6) has summarized the evidence for an important function of monocytes in certain cases of virus-induced inhibition of lymphocyte transformation. For example, it is clear that monocytes infected with polio virus cannot perform their essential helper function (2) on lymphocyte transformation. Comparable results have been described for the role of adhering cells in the effect of herpes simplex virus on mouse lymphocyte cultures (3). Effects of rubella virus (14) and murine cytomegalo virus ( 15) on lymphocyte stimulation, however, could not be attributed to monocyte inactivation. Sullivan et al. reported that cultures of leukocytes, depleted monocytes, could still be inhibited by measles virus (16). This was confirmed in experiments performed in this laboratory with pure T cells after sedimentation of E rosettes from monocyte-depleted suspensions. Such data, however,

MEASLES

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FUNCTION

4.57

are not conclusive because the few, and essential, monocytes still present might become infected and thus not perform the right activity. Therefore, we designed experiments in which noninfected monocytes could be added under conditions which made sure that they remained noninfected. As shown, mixed lymphocyte reactions and the stimulation with Con A under these conditions were still inhibited when the responding cells had previously been infected with measles virus, indicating that irradiated accessory cells are not able to counteract the effect of measles virus on lymphocyte responsiveness in vitro. We have tried to infect monocytes with measles virus so that we could add infected monocytes to noninfected monocyte-depleted lymphocyte cultures. It proved difficult to infect monocytes to a significant extent, which made the experiment suggested above technically impossible, and results are not shown. We observed that the stimulatory capacity of lymphocytes in the MLR was diminished considerably after infection with measles virus. In view of the results of experiments in which infected and noninfected lymphocytes were mixed in the presence of human serum containing antibodies against measles virus, the possibility that measles virus leaks from the irradiated stimulator cells and infects the responding cells is excluded. It is interesting to note that a lymphocyte function that does not require DNA synthesis is inhibited in virus-infected cells. This is, as far as we know, the first example to be described. No information as to the mechanism by which measles virus affects the stimulatory capacity of lymphocytes in the MLR is available. We have recently found that lymphocytes that had been infected with measles virus were, after irradiation, still capable of expressing measles virus antigens upon stimulation. It is very likely that infected, irradiated stimulator cells in the MLR are stimulated by the alloantigens on the responding cells. This should lead to increased viral activity in these cells which, in some way, apparently interferes with their stimulating capacity. It is well known that only intact lymphocytes can serve as stimulator cells in a primary MLR (17). DNA synthesis is not required in the stimulating cell, although conflicting data exist as to whether protein synthesis is required (18). Experiments are in progress on the analysis of the effects of measles virus on cellular protein synthesis in stimulated lymphocytes. Haspel et al. failed to show an inhibition of protein synthesis by measles virus (19). Apart from the idea that there is an influence of measles virus on metabolic processes required in the stimulator cell in a MLR, it might be postulated that this inhibition induced by measles virus is caused by the alteration of cell surface determinants, although Haspel et al. found no influence of measles virus on the expression of HLA-A and B antigens (19). Since only B lymphocytes can serve as stimulator cells in the MLR (20), we conclude that apparently B lymphocytes can become infected with measles virus. This means that both B- and T-lymphocyte functions can be inhibited by infection with measles virus in vitro. In the results shown in this study, the inhibition of PHA stimulation was always greater than the inhibition of Con A stimulation and the inhibition of the responding cell in the MLR. This is in contrast with results published previously where measles virus was added together with, or shortly after, the stimulating agent or cell. We have no adequate explanation for this difference. One might speculate that the presence of PHA enhances cell fusion (21) thereby facilitating spreading of the infection via cytoplasmic bridges, which make the virus inaccessible

4.58

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for extracellular antibodies. However, this is not in agreement with results obtained with the experiments in which infected and noninfected lymphocytes were mixed and cultured with PHA (Table 5). On the other hand, it is known that Con A interferes with the replication of enveloped viruses (22). Recently, we have investigated the killing capacity of lymphocytes infected with measles virus. The results (to be published in a forthcoming paper) show that the activity of K lymphocytes in killing sensitized target cells in the ADCC is not impaired after infection of lymphocytes with measles virus. ACKNOWLEDGMENTS We should like to mention discussions with Drs. A. C. Allison, V. P. Eijsvoogel, C. J. M. Melief, P. Th. A. Schellekens, and W. P. Zeijlemaker. We are grateful to Dr. Billiau and his co-workers for their kindness in performing the interferon titrations.

REFERENCES 1. Lucas, C. J., Galama, J. M. D., and Ubels-Postma, J. C., Cell Zmmunol. 32, 70, 1977. 2. Soontiens, F. J. C. S., and Veen, J van der, J. Znzmunol. 111, 1411, 1973. 3. Kirchner, H., Darai, G., Keyssner, K., Munk, K., and Mergenhagen, S. E., In “Regulatory (D. 0. Lucas, Ed.), pp. 617-619. Academic Mechanisms in Lymphocyte Activation” Press, New York, 1977. 4. Oers, M. H. J. van, Pinkster, J., and Zeijlemaker, W. P., Znt. Arch. Allerg. A#. Zmmunol., in press. 5. Alter, B. J., and Bach, F. H., Cell. Znzmunol. 1, 207, 1970. 6. Virelizier, J. L., Biomedicine 22, 255, 1975. 7. Blomgren, H., Strander, H., and Cantell, K., Stand. J. Znzmunol. 3,677, 1974. 8. Notkins, A. L., In “Viral Immunology and Immunopathology” (A. L. Notkins, Ed.), pp. 149-166. Academic Press, New York, 1975. 9. Billiau, A., Edy, V. G., Heremans, H., Damme, J. Van, Desmijter, J., Georgiades, J. A., and Somer, P. de, Antimicrob. Ag. Chemother., in press. 10. Lucas, C. J., Ubels-Postma, J. C., and Galama, J. M. D., Manuscript submitted for publication. 11. Klimpel, G. R., Dean, J. H., Day, K. O., Chen, P. B., and Lucas, D. O., Cell. Zmmunol. 32, 293, 1977.

12. Wheelock E. F., and Toy, S. T., Advun. Znzmunol. 16, 123, 1973. 13. Heron, I., Berg, K., and Cantell, K., J. Zmmunol. 117, 1370, 1976. 14. Logt, J. T. M. van der, Thesis, Nijmegen, The Netherlands. 15. Selgrade, M., Ahmed, A., Sell, K. W., Gershwin, M. E., and Steinberg, A.D., J. Zmmunol. 116, 1459, 1976. 16. Sullivan, J. L., Barry, D. W., Albrecht, P., and Lucas, S. J., J. Zmmunol. 114, 1458, 1975. 17. Wagner, H., Eur. I. Zmmunol. 3, 84, 1973 18. Metzler, C. M., Koslyk, T. G., and Gershon, R. K., J. Zmmunol. 117, 1295, 1976. 19. Haspel, M. V., Pellegrino, M. A., Lampert, P. W., and Oldstone, M. B. A., J. Exfi. Med. 146, 146, 1977. 20. Oers, M. H. J. van, and Zeijlemaker, W. P., Cell. Zmmunol. 31, 205, 1977. 21. Poste, G., Alexander, D. J., Reeve, P., and Hewlett, G., J. Gen. Viral. 23, 255, 1974. 22. Okada, Y., and Kim, J., Virology 50, 507, 1972.