Host defense mechanisms against infectious bovine rhinotracheitis virus

CELLULARIMMUNOLOGY17, 43--56 (1975)

Host Defense Mechanisms Against Infectious Bovine Rhinotracheitis Virus II. Inhibition of Viral Plaque Formation by Immune Peripheral Blood I_ymphocytes1 BARRY

T. ROUSE

AND

LORNE A. BABIUK

Department of Veteri~ary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Canada S7N OWO Received July 8, 1974 A method is described to measure in vitro the activity of bovine peripheral blood lymphocytes (PBL) to inhibit viral plaque formation by a herpes virus common to cattle, named infectious bovine rhinotracheitis (IBR) virus. Microtitre plates containing 96 Madin-Darby bovine kidney cell monolayers were infected with a range of concentrations of IBR virus, and PBL from immunized or normal control animals were added to the monolayers. Viral plaque formation (both plaque number and size) was markedly inhibited by PBL from immune animals but not by PBL from normal controls. Immune (to IBR) PBL failed to inhibit Herpes simplex virus. The mechanism of inhibition would appear to involve a suppression of viral replication rather than destruction of virus, since on removal of immune PBL from monolayers, viral plaques appeared. The importance of the assay as a quantitative measure of cell-mediated immunity against a viral antigen is emphasized. INTRODUCTION It is now abundantly clear that the specific immune response to antigen is protean, involving humoral and cellular components which can act together in a protective way or which act opposingly with one component suppressing another ( 1 - 4 ) . T h e respective importance and interplay of the various aspects of the immune response has not been adequately explored in the case of viral agents, yet a full understanding is necessary if existing and future immunoprophylactie measures are to be optimally employed. The role of various immunoglobulins in resistance to and recovery from viral infections has been well investigated, especially regarding their protective role ( 5 - 8 ) . T h e part played by cell-mediated immune mechanisms is less understood and has only recently received much attention ( 9 18). I n the present paper we describe a simple reproducible assay that quantitates cell-mediated immunity ( C M I ) 2 against a herpes virus--infectious bovine rhinotraeheitis ( I B R ) virus. T h e assay quantitates the activity of lymphocytes, immune to I B R antigen, to inhibit the cytopathic effects of I B R virus plaques in cell monolayers. 1 Supported by Grant MA 5067 of the ~edical Research Council of Canada. e Abbreviations used in this paper: CMI, cell-mediated immunity; MDBK, Madin-Darby bovine kidney cells; IBR, infectious bovine rhinotracheitis virus; MEIv[, Eagle's base minimum essential medium; FCS, fetal calf serum; PFU, plaque-forming units; ERP, equine rhinopneumonitis virus; PS Puck's solution G; PBL, peripheral blood lymphocytes. 43 Copyright © 1975 by AcademicPress, Inc. All rights of reproductionin any form reserved.

44

ROUSE AND BABIUK

MATERIALS AND METHODS

Cells Madin-Darby bovine kidney (MDBK) cells were obtained from International Scientific Industries and were free of mycoplasmas at the time of reception. The cells were cultured in Eagle's base minimal essential medium (MEM) containing 10% heat-inactivated fetal calf serum (FCS). Each litre of medium was supplemented with 10 ml nonessential amino acids (Gibco, Cat. No. 114), glutamine (2 mmoles), 100 mg kanamycin, and 2.5 g of sodium bicarbonate.

Virus Infectious bovine rhinotracheitis (IBR) virus, strain P8-2, was isolated at Purdue University by Dr. J. R. Saunders. This virus received two passages through primary fetal bovine kidney monolayers prior to preparation of stock virus. Stock virus was prepared by infecting roller bottles of MDBK cells. Virus (0.1 PFU/cell) was allowed to adsorb for 1 hr at 37°C, the unadsorbed virus removed, and MEM containing 4% heat-inactivated FCS added. After incubation at 37°C for 48-72 hr, all cells were rounded and approximately 90% were detached. The remaining attached cells were removed from the glass, and the entire culture fluids and cells were subjected to two freeze-thaw cycles. Cellular debris was removed by centrifugation at 1000 9 for 10 min, after which the virus-containing supernate was dispensed into small vials and stored at -70°C. This constituted the stock virus. Repeated titration of virus in MDBK cells gave a titre of 1.1 X l0 s PFU/ml.

Herpes simplex Herpes simplex type 1 was obtained from the ATCC by way of the University of Saskatchewan, Department of Bacteriology. This virus was passed twice through MDBK cells, after which cytopathic effects were visible. The fourth MDBK cell passage was used as stock virus and was prepared essentially as described above. In MDBK cells, the H. simplex stock virus had a titre of 1.1 × 10~ PFU/ml. Plaque Inhibition Assay Falcon plastic tissue culture plates (No. 3040) containing 96 wells (6 ram) were seeded with 50 × 103 MDBK cells per well. After 18 hr, when monolayers were confluent, cells were infected with varying concentrations of IBR virus. Following adsorption for 1 hr at 37°C in a humidified CO~ (5~o) incubator, the nnadsorbed virus was removed and either culture medium (virus controls) or culture medium containing varying numbers of lymphocytes was added to the wells. In most experiments the culture medium was MEM containing 4% heat-inactivated FCS and 2 neutralizing units of bovine anti-IBR antisera (i.e., serum with a neutralization titre of 1/320 was used at a final concentration of 1/160). The culture plates were incubated for 3 days, and the fluids and nonadherent cells were removed by vigorously inverting the plate. The remaining nonadherent cells were removed by gently washing in saline, after which the monolayers were fixed and stained with 1% gentian violet in 70% ethanol. Excess dye was removed by washing in distilled water, the plates dried, and the results were recorded by magnification using an

OF VIRAL PLAQUES BY IMMUNE

INHIBITION

LYMPHOCYTES

45

overhead projector. The diameter of individual plaques was measured with a micrometer, and the average plaque and area was computed. To facilitate an appreciation of the inhibitory effects of immune lymphocytes, the results were also expressed as the product of the number of plaques (N) and plaque area (A). Using these values, the percent inhibition was computed according to the following formula : % inhibition =

N )< A virus control - N X A with PBL N )< A virus control ;< 100.

Assays were performed in duplicate or quadruplicate and experiments were repeated several times.

Preparation of Peripheral Blood Lymphocytes (PBL) Blood was collected by venipuncture into a syringe containing preservative-free heparin (5 IU/ml of blood collected). The buffy coat was obtained after centrifugation at 800 g for 20 min at 4°C, and these cells were diluted in Puck's solution G (PS) (buffy coat of 35 ml of blood diluted to 10 ml). These leucocyte rich cells were layered onto a 3-ml volume of Ficoll-Hypaque (density at 25°C 1.077 g/cm3 : Ficoll, Pharmacia; I-typaque, sodium diatrizoate, Winthrop Labs.) in a 13 × 120mm centrifuge tube. Following centrifugation at 400 g for 20 rain at room temperature, the lymphocyte-enriched cells were collected from the interface of plasma and Ficoll-Itypaque. These cells were washed once with PS, the red cells lysed with 0.83~ ammonium chloride (5 rain at 37°C) and then washed twice prior to enumeration. Cells harvested by this technique were invariably more than 98 9 viable and consisted of > 9 9 ~ mononuclear cells. Known numbers of cells were suspended in MEM containing 4% FCS (and antisera in most experiments) and were added in a 0.2-ml volume to wells in the culture plate.

Immunization Procedures Young adult steers were immunized intramuscularly on three occasions at monthly intervals with 109 PFU of IBR virus. On the first injection the virus was emulsified in Freund's complete adjuvant. These animals were repeatedly bled after the second and third immunizing dose, and both their lymphoeytes and sera were used in the experiments described subsequently. Anti-H. simplex antisera was obtained from a human volunteer who had recently recovered from a labial herpetic lesion.

Normal Control Animals Adult cows with no history or serological evidence of exposure to IBR and young calves were used as a source of normal lymphoeytes. Some of these animals were immunized with Freund's complete adjuvant, but this did not change the reactivity of their lymphocytes against IBR virus plaques.

Treatment of Lymphocytes with Mitomycin C Mitomycin C (Calbiochem, La Jolla, CA) was diluted to contain 250 /~g/ml and stored at -70°C. For treatment of lymphocytes, 107 cells in 0.9 ml were

46

ROUSE AND BABIUK

treated at 37°C for 30 rain with 0.1 ml mitomycin C (25 ~g). The treatment prevented cell division as demonstrated by the inability of treated lymphocytes to respond to an optimum stimulating dose of phytohemagglutinin ( P H A ) (Burroughs Wellcome, Beckenham, England). RESULTS

Inhibition of Viral Plaques by Immune Ly~phocytes To determine if P B L from immunized animals were inhibitory to viral plaque production, monolayers of M D B K cells were infected with a range (2-2 × 10 * P F U ) of concentrations of I B R virus, and then varying numbers of immune or normal lymphocytes were added to the monolayers. Following 3 days of culture, monolayers were examined to enumerate plaques and measure their area. The results of a representative experiment, in which the activity of immune and normal P B L were compared, are shown in Fig. 1. As can be seen, plaques in virus control monolayers and in monolayers that received normal P B L were distinctly visible, whereas a marked inhibition of plaque development was apparent in monolayers that received 10 6 immune PBL. In this experiment, low levels (2 neutralizing Units) of bovine anti-IBR serum were used since this facilitated plaque enumeration, especially at higher levels of virus. However, not shown in Fig. 1, inhibition

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FIG. 1. Effect of varying numbers of PBL from immune and normal animals on IBR virus plaques in MDBK monolayers. With the exception of the cell controls (lower right) monolayers were infected with one of a range of virus concentrations. Following adsorption for 1 hr at 37°C, varying numbers of Iymphocytes suspended in culture medium containing 4% FCS and 2 neutralizing units of antibody were added. After 3 days of culture, monolayers were washed to remove dead and nonadherent cells, fixed, stained, and photographed.

INHIBITION OF VIRAL PLAQUES BY IMMUNE LYMPHOCYTES

47

of the same magnitude occurred when antiserum was not incorporated into the medium. In Table 1, the results of a representative experiment (repeated more than 10 times) in which PBL from an animal immunized on three occasions with IBR virus are compared with PBL from two nonimmunized control animals. Immune PBL inhibited both plaque number and plaque size, especially the latter. Thus, 106 immune PBL completely prevented the development of visible plaques in monolayers that received 200 P F U of virus or less. With 2 × 10~ PBL, plaque numbers were not reduced in monolayers that received 200 P F U of virus but a marked reduction in plaque area was apparent. The percent inhibition in this instance was 60~'o. Fifty thousand immune PBL were not inhibitory. It should be noted in this system that 200 P F U do not give 200 visible plaques. This is a common observation with viral plaque development (19). Whereas immune PBL caused inhibition of both plaque number and area, normal PBL failed to do either. If more than 10" PBL, either immune or normal, were added to monolayers, then the lymphocytes were usually toxic to the monolayers. If the lymphoeytes were not toxic, plaque development usually did not occur, both in the case of immune and normal PBL, indicating that inhibition in these circumstances was nonspecific. As mentioned above, similar results were obtained using serum without specific antibody in the culture fluids. However, it was more difficult to discern plaques especially at the higher virus concentrations. That inhibition, nonetheless, occurred with immune PBL but not with normal PBL could be demonstrated by measuring the yield of virus from monolayers at the end of the 3-day culture period. The results of two such experiments are expressed in Table 2. It can readily be seen that immune PBL markedly reduced virus yields whereas normal PBL failed to do so. Thus, 108 immune PBL reduced the virus yield by 99.9%, and even 2.5 × 10~ PBL gave some reduction in virus yield (73}5 in one experiment).

Failure of IBR Virus to Replicate in Bovine Lymphocytes Because it was often noticed that plaques were larger and sometimes more numerous in monolayers with an overlay of normal PBL as compared to monolayers without any PBL, experiments were performed to investigate whether or not virus could replicate in either normal or immune PBL. The respective PBL were seeded into petri dishes and known concentrations of IBR virus were added. Control petri dishes containing culture media were seeded with the same concentrations of virus. At various time intervals aliquots of culture fluids were taken, the cells removed, and ttle titre of virus determined. As can be seen in Fig. 2, no difference in virus titre between the three groups was apparent, indicating that viral replication had probably not occurred in either the normal or the immune PBL. Experiments to prove further that viral replication failed to occur are in progress. The results expressed in Fig. 2 also indicate that neither immune nor normal PBL appear to inactivate virus.

Effect of Removal of Lymphocytes from Infected Monolayers Although immune PBL can inhibit virus, apparently they do not completely prevent viral replication. Thus, as indicated in Table 2, low levels of virus could still be recovered from monolayers that showed no sign of cytopathology~ Results,

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FIG. 2. Failure of IBR virus to replicate in normal or immune bovine PBL. Three petri dishes received either RPMI 1640 plus 10% FCS, 2 × 106 immune PBL in RPMI 1640 plus 10% FCS, or 2 × 10~ normal PBL in RPM 1640 plus 10'% FCS. All nine dishes were seeded with 108 PFU of IBR virus at time 0, and at daily intervals the virus titre in an aliquot of culture fluids was determined. The points represent the mean --- SE titres without PBL (O--O), with immune PBL (A--A), and with normal PBL ( × - - × ) . subsequently to be reported (20), have shown that the low titre of virus recovered after 3 days of culture does not entirely represent residual virus. Evidence that immune Iymphocytes do not destroy virus in infected monolayers is given in Table 3. In the experiment, infected monolayers were overlayed with immune lymphocytes. At intervals afterwards, the lymphocytes were removed, the monolayers washed, and antibody-containing medium added. The results were recorded after a further period of incubation. Removal of immune P B L at 24 hr failed to inhibit viral plaques as measured after a further 2 days of culture. However, if the P B L were left in contact with the virus-infected monolayers for 48 hr, some inhibition (7384~o) was apparent on examination the following day. Even if immune P B L were left on the monolayers for as long as 3 days prior to removal, plaques still appeared if the monolayers were held for a further 2 days. These experiments could indicate that lymphocyte inhibition is a suppression of virus replication rather than a destruction of virus and virus-infected cells.

Specificity of Plaque Inhibition Assay Two approaches were used to examine for specificity of plaque inhibition. In the first approach, animals known not to have been exposed previously to I B R virus and whose P B L failed to inhibit viral plaques were infected intranasally with I B R virus and then sampled periodically over the ensuing 3 weeks. Figure 3 illustrates the time after infections when P B L develop the ability to inhibit viral plaques in vitro. In this series of experiments, M D B K cultures were infected with 100 P F U of virus and overlayed with 106 PBL. As can be seen, inhibition was first detectable on day 3 (68.5~o), was at peak activity on day 5 (98.4%), and declined thereafter.

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Definite inhibition was still detectable on day 24 (the latest day of examination). Lymphocytes from 3× immunized animals retained their inhibitory activity for many weeks. TABLE 4 SPECIFICITY OF PLAQUE INHIBITION BY NORMAL AND IBR IMMUNE BOVINE LYMPHOCYTES AGAINST IBR, AND H. SIMPLEX VIRUSESa

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53

I N H I B I T I O N OF VIRAL PLAQUES BY IM1VLUNE LY1ViPI-IOCYTES

TABLE 5 INHIBITION OF I B R VIRUS PLAQUES BY IMMUNE BOVINE LYMPHOCYTES

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PBL from an animal 3;< immunized with IBR virus. b Cells held at 56°C for 30 min. Cells were 93% dead as judged by trypan blue exclusion. c Lymphocytes (106) treated with 25 ug-1 mitomycin at 37°C for 30 rain, 1 ml volume. Washed cells were unable to respond to an optimal stimulating dose of PHA. e Number of visible viral plaques. e Percent inhibition determined as described in Table 1'. * Statistically significant at +0.05% level from the virus control. The second approach to check for specificity was to determine the inhibitory effect of immune (to I B R virus) and normal P B L against another herpes virus. As shown in Table 4, immune but not normal P B L inhibited I B R viral plaques. Against H. simplex, 106 P B L from both normal and immune animals produced some inhibition against lower levels of virus inoculation. Lower cell numbers were not inhibitory.

Requirements for Viable Cells Shown in Table 5 are data indicating that the immune P B L cells must be viable in order to be inhibitory; thus, heating cells to 56°C for 0.5 hr, after which time cells were 90-95% dead as judged by trypan blue exclusion, removed the inhibitory effect. Similarly, it would appear that lymphocytes must be capable of dividing if they are to have inhibitory activity, since cells treated with mitomycin C failed to cause inhibition of viral plaques. DISCUSSION Numerous proposals have been made to explain the mechanisms of resistance to and recovery from virus infections (6-12, 15, 16). On infection, the host usually mounts both a humoral and cell-mediated immune response as well as many nonspecific protective effects. The importance of any one particular immunological mechanism probably varies with the nature of the virus-host cell relationship. Thus, in the case of herpes viruses, which spread from infected cells to susceptible cells by intercellular bridges (21), the virus need not enter the extracellular environment of the cell. Consequently, humoraI antibody may play little, if any, part in the recovery process from herpes viruses (22, 23). Apparently under some circumstances antibody, along with complement, can destroy a virus-infected cell but this mechanism is not effective until after the virus has already spread to contiguous cells (15). Many have suggested that cellular defense mechanisms may be those most important for defense against herpes viruses, but documentation of this notion is sparse because of the lack of suitable in vitro assays to quantitate C M I adequately against viruses.

54

ROUSE AND BABIUK

We have described a simple and reproducible assay that can be used to quantitate CMI against a herpes virus common to cattle--infectious bovine rhinotracheitis (IBR) virus. Using microtitre plates containing 96 cells monolayers, PBL of specifically immunized animals were shown to prevent the cytopathic effect of IBR virus. The advantage of such a microtitre system is that in one experiment it is feasible to determine the degree of inhibition by varying numbers of lymphocytes against a wide range of virus concentrations. The plates can easily be stored and visualized by projection on a screen. Whereas immune lymphoeytes could inhibit the cytopathic effects of 2 × 104 P F U of virus, the same number of lymphocytes from nonimmunized animals were not appreciably inhibitory even to 2 P F U of virus. However, if 2 × 106 or more lymphocytes, either immune or normal, were added to virus-infected monolayers, then such cells were usually toxic; the monolayer was destroyed either partially or completely. Under these circumstances even normal lymphocytes inhibited plaque formation. Presumably, large numbers of lymphocytes render monolayers refractory to virus perhaps by releasing toxic products that affect cellular metabolism. A number of approaches were used to demonstrate that inhibition in the system was immunologically specific, thereby giving support to the hypothesis that the in vitro assay is a measure of a specific cell-mediated defense mechanism. The fact that only immune cells were inhibitory was mentioned above. In addition, it was clearly apparent that lymphocytes acquired the capacity to inhibit virus plaques after infection of animals with IBR virus. This inhibitory activity had declined by 3 weeks after infection. Specificity was also demonstrated by experiments showing that lymphocytes from animals immune to IBR virus were not enhanced in their inhibitory activity against another herpes virus H. simplex. In the assay described, low levels of neutralizing antibody were routinely used to facilitate plaque visualization. Accordingly, it could be implied that immune lymphocytes could not suppress virus cytopathology but that lymphocytes must interact with humoral antibody to do this. Such a mechanism has been suggested by Ennis (16) in the case of rabbit spleen cell inhibition against H. simplex virus. However, we have demonstrated that, even in the absence of antibody, inhibition still occurs. This was documented by showing that immune lymphocytes could reduce virus yield by 99.9% whereas normal lymphocytes were barely inhibitory. How immune lymphocytes can inhibit virus replication remains to be defined. In confirmation of previous findings (15), it appears that cells must be viable. They must also be capable of division since mitomycin C-treated cells were not inhibitory. Furthermore, inhibition of virus replication is not absolute since, although visible signs of virus eytopathology may be completely prevented, some virus replication must occur because viral plaques appear after the removal of lymphocytes from the monolayer. In addition, neither immune nor normal lymphocytes per se inactivate free virus. This could mean that activated lymphocytes release some factor that renders susceptible cells refractory to viral infection so that virus cannot spread. This notion was supported by the observation that inhibition was more evident at the level of plaque size that number. Additional factors may suppress virus replication in infected cells. Whether or not interferon, a factor that can be released from lymphocytes upon antigenic stimulation (12, 24), is involved in the resistance mechanism is under investigation. However, because herpes viruses are relatively insensitive to interferon (25, 26), it may not play a major role in the mechanism of resistance.

INHIBITION

OF

VIRAL PLAQUESBY IMMUNE

LYMPHOCYTES

55

An alternative mechanism is that immune lymphocytes recognize and react with infected cells since such have viral antigen on their surface (27). The result of this recognition is destruction of the infected cells. This latter explanation seems unlikely since it has been shown that virus can spread to new cells before they become susceptible to lymphocyte destruction (15). Second, and perhaps more important, complete or partial replication by a cell of a herpes virus is invariably followed by the death of the cell (28). Consequently, the destruction of the virusinfected cell by a lymphocyte (or by some other means such as antibody and complement) would not seem to be beneficial to the host if the cell is destined to die anyhow. We favor the hypothesis that the mechanism of inhibition involves the release of soluble products, perhaps including interferon, that protects noninfected cells from infection and which may, in addition, suppress virus replication in infected cells. Experimental support for such a hypothesis is under investigation. Repeatedly in this discussion we have implied that the inhibitory effect is mediated by lymphocytes, and not another cell type such as a macrophage. Previous studies using a similar assay system have suggested that the inhibitory cell is a macrophage not a lymphocyte (15) and, furthermore, that nonspecifically activated macrophages were even more inhibitory. In contrast, other data have indicated that inhibition is mediated by a lymphocyte (16). Presumably, in the body both cell types could interact to defend against herpes viruses. The system we have described should prove useful to help unravel the nature of these cell interactions. ACKNOWLEDGMENTS Our appreciation to Lynn Franson for enthusiastic and competent technical assistance, to Dave Geary for the line diagrams, and to Dr. S. Federoff and Dr. C. Bigland for critically reviewing the manuscript. REFERENCES 1. 2. 3. 4. 5. 6.

Katz, D. H., and Benaceraff, B., Advan. Immunol. 15, 1, 1972. Miller, J. F. A. P., Int. Rev. Cytol. 33, 77, 1973. Allison, A. C., Arthritis Rheum. 32, 283, 1973. Herzenberg, L. A., and Ylerzenberg, L. A., Contemp. Top. Immunobiol. 3, 41, 1974. Tomasi, T. B., and Bienenstock, J., Advan. Immunol. 9, 1, 1968. Waldmann, R. H., Kasel, J. A., Alford, R. H., and Mann, J. J., Proe. Soe. Exp. Biol. Med. 125, 316, 1967. 7. Allison, A. C., in "The Scientific Basis o~ Medicine" (I. Gilliland and J. Francis, Eds.), pp. 49-73. Humanities Press Inc., New Jersey, 1972. 8. Rouse, B. T., and Ditchfield, W. J. B., Res. Vet. Sci. 11, 503, 1970. 9. "Cell Mediated Immunity and Resistance to Infection." WHO Tech. Report Series No. 519, 1973. 10. Allison, A. C., Ann. Inst. Pasteur 123, 585, 1973. 11. Blanden, R. V., J. Exp. Med. 133, 1090, 1971. 12. Glasgow, L. A., Arch. Intern. Med. 1216, 125, 1970. 13. Speel, L. F., Osborn, J. E., and Walker, D. L., d-. Immunol. 101, 408, 1968. 14. Steele, R. W., Hensen, S. A., Vincent, M. M., Fuccillo, D. A., and Bellanti, J. A., J. Immunol. 110, 1502, 1973. 15. Lodmell, D. L., Niwa, A., Hayashl, K., and Notkins, A. L., J. E.~:p. Med. 137, 706, 1973. 16. Ennis, F. A., Infect. Immunol. 7, 898, 1973. 17. Rabinowitz, S. G., and Proctor, R. A., J. Immunol. 112, 1070; 1974. 18. Rouse, B. T., and Babiuk, L. A., Infect. Immunol. 10, 681, 1974. 19. Thomas, F. W., Ph.D. thesis, University of Wisconsin, 1972. 20. Babiuk, L. A., and Rouse, B. T., manuscript in preparation.

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21. Christion, R. T. and Ludovici, P. P., Proc. Soc. Exp. Biol. Med. 138, 119, 1971. 22. Centifanto, Y. M., Little, J. M., and Kau{man, It. E., Ann. N. Y. Acad. Sci. 173, 649, 1970. 23. Douglas, R G., and Couch, R. B., J. Immunol. 104, 289, 1970. 24. Merigan, T. C., New Engl. J. Med. 290, 323, 1974. 25. Glasgow, L. A., Hanshaw, J. B., Merigan, T. C., and Petralli, J. K., Proc. Soc. Exp. Biol. Med. 125, 843, 1967. 26. Barahona, H. H., and Melendez, L. V., Proc. Soc. Exp. Biol. Med. 136, 1163, 1971. 27. Roane, P. R., and Roizman, B., [Zirolo9y. 22, 1, 1964. 28. Roizman, B., in "Oncogenesis and Herpes Viruses" (P. M. Biggs, G. de The, and L. N. Payne, Eds.), pp. 1-17. International Agency for Cancer, Lyon, 1972.