INFECTION AND IMMUNITY, Mar. 1980, p. 767-776 Vol. 27, No. 30019-9567/80/03-0767/10$02.00/0

Role of Specific Cytotoxic Lymphocytes in Cellular ImmunityAgainst Murine Cytomegalovirus

MONTO HOJohn Curtin School ofMedical Research, Canberra, Australia, and Graduate School ofPublic Health and

School ofMedicine, University ofPittsburgh, Pittsburgh, Pennsylvania 15261*

Cytotoxic lymphocytes were generated in vitro against murine cytomegalovirus(MCMV)-infected cells by incubation with ultraviolet light-irradiated, infectedfibroblasts. When passively transferred, they reduced virus titers in spleens ofmice 1 day after infection with MCMV. Protection was abrogated by anti-@ serumand complement. Spleen cells from mice infected for 6 to 14 days protected micebetter than cells from mice after infection for 1, 3, or 30 days. Protection by invitro- and in vivo-generated cells was H-2K or H-2D restricted. Specific cytotoxicT lymphocytes are therefore present and operative during acuteMCMV infection.However, MCMV infection inhibited the development of primary cytotoxicresponse against ectromelia virus. It also suppressed the ability of lymphocytesfrom mice with established memory for ectromelia to develop secondary cytotoxiccells in vitro, and it inhibited the development of memory cells for the cytotoxicresponse to ectromelia virus. In view of these data and the inability of animalsrecovering from MCMV infection to eliminate all infected cells, the cytotoxicresponse to MCMV may be qualitatively or quantitatively deficient.

Besides acute infections, the herpesviruses in-cluding cytomegaloviruses produce persistent la-tent infections which reactivate at unpredictableintervals or when the host is inmunosuppressed(13, 25). Cytomegalovirus infections are partic-ularly common when cell-mediated immunity issuppressed (12). Although it is assumed thatcell-mediated immunity is important in host de-fenses against cytomegaloviruses, the elucida-tion of this type of immunity, even in the muchstudied mouse model, has been slow.We now know that passively transferred im-

mune T lymphocytes protect mice from deathcaused by marine cytomegalovirus (MCMV) in-fection (36). T lymphocytes from immune miceproliferate in the presence of cell-associated (20)and free MCMV (15). The development of H-2K or H-2D restricted cytotoxic T lymphocytes(CTL) in infected animals, which has been de-scribed for many other viruses (5; R. M. Zink-ernagel and P. C. Doherty, Adv. Immunol., inpress), has lately been described for herpes sim-plex virus and for MCMV (33, 34). Ho andAshman (14) also described recently the in vitrogeneration of CTL against MCMV.

In classical studies on the cell-mediated im-mune resistance against ectromelia virus, Blan-den showed that only T lymphocytes from re-cently (4 to 20 days) infected animals were pro-tective when passively transferred (4). The abil-ity of effector spleen lymphocytes to protectagainst virus replication and to lyse infected

targets coincided when compared over 10 daysafter infection, and protection was H-2K or H-2D restricted (7,18). B lymphocytes did not playa protective role in these passive transfer exper-iments (6). The assumption is that CTL wereresponsible for the passive protection, and thatthey constitute the mainstay of immune defen-ses early in the course of infections (Zinkernageland Doherty, in press). This assumption isstrengthened by the finding that secondary cy-totoxic cells developed in vitro against ectro-melia and influenza viruses are also protective(19, 37). The precise place of CTL relative toother specific and nonspecific defense factors,such as lymphokines, interferon, natural killercells, and even early antibody formation (2), andthe protective role of T cells in chronic virusinfection (M. B. C. Dunlop, Ph.D. thesis, Aus-tralian National University, 1977), has been lesswell studied.

Since MCMV differs from other viruses whichproduce only acute disease, one might askwhether there is a defect in the cytotoxic re-sponse to this virus so that infected cells escapedestruction. If the response is present, it is ac-tually functional? The following report attemptsto answer these queries.

MATERIALS AND METHODSMice. The following strains of mice were obtained

from the breeding colony of the John Curtin School ofMedical Research and used as young adults 5 to 10

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weeks of age: BALB/c (H-2d), A.TL (Ka, Ik, Dd), CBA/H (H-.2), SJL/J (H-Z), and C57BL/6J (H2b). Whenthe identity of strain is not specified, BALB/c micewere used.

Viruses. The Smith strain of MCMV was obtainedfrom June Osborn. The salivary gland virus (SGV)stock was made from pooled salivary glands ofBALB/c mice infected for 4 to 5 weeks. It titered 1066 to 1068plaque-forming units (PFU)/1.0 ml. When unspecified,this stock was used. To obtain tissue culture-passedvirus (TCV), SGV was diluted to 1:100 and inoculatedin a culture of mouse embryo fibroblasts (MEF). Themedium was harvested when 4+ cytopathology wasobserved, which was usually 4 to 5 days after inocula-tion. This harvest was serially passed in cell cultureseight times. The final harvest (1:50 dilution) was usedto inoculate a confluent bottle culture of MEF with asurface area of 360 cm2 and containing 50 ml of me-dium. The culture was incubated, and rolled and har-vested 5 days later. This stock titered 1070 PFU/1.0ml.The attenuated (Hampstead) egg passage strain of

ectromelia virus as well as the virulent (Moscow)mouse spleen-passaged virus was used. The method ofgrowing, titrating, and storing these strains is de-scribed in Gardner et al. (11).Memory mice. This term refers to mice which were

primed by infection with ectromelia virus or MCMV4 to 8 weeks before use. Mice were injected intrave-nously (i.v.) with 2 x 104 PFU ofattenuated ectromeliavirus or with 104 PFU of MCMV intraperitoneally(i.p.).

Cell culture and medium. MEF cultures wereprepared from embryos of 16- to 18-day-pregnantBALB/c or other strains as previously described (39).They were grown in Eagle minimal essential mediumsupplemented by 10% inactivated calf serum, gluta-mine, antibiotics, and bicarbonate. Primary culturesconsisted of 107 cells in 30 ml in 75-cm2 flasks (FalconPlastics, Oxnard, Calif.). For preparation of secondarycultures, a trypsinized suspension of 2 x 105 cells perml in medium was made from a primary culture.Multiwell (24- and 96-well) flat-bottomed microcultureplates (Linbro Scientific, Inc., Hamden, Conn.) eachreceived 1.0 or 0.1 ml per well. All cultures wereconfluent 24 to 48 h after initiation and used within 1week. All incubation was at 370C in a 10% humidifiedCO2 atmosphere.When lymphocytes were cultured to measure their

proliferative response or for the development of cyto-toxic cells, the medium (lymphocyte medium) wassupplemented with 10-4 M 2-mercaptoethanol and10% fetal calf serum (Commonwealth Serum Labora-tory, Melbourne, Australia) prescreened for cellgrowth-promoting property.

Collection of spleen cells. Spleens were collectedfrom mice in 10 ml of Puck A saline (0.8% NaCl, 0.4%KCl, 1% glucose, 0.035% NaHCO3, 0.002% phenol redin deionized water) and pressed through a stainless-steel wire screen. Cell clumps were dissociated bypipetting, and fibrous material was removed by quickcentrifugation for 30 s. The cell preparations werewashed two times in lymphocyte medium and counted.UV and y-irradiation. Before ultraviolet light

(UV) irradiation, MEF monolayers were covered with

INFECT. IMMUN.

medium to about 1 mm in depth. A 30-W germicidallamp which emitted in the range of 230 to 270 nm wasapplied for 4 min (22). Samples were placed at adistance which produced 960 jtW/cm2 measured by aUV meter (UV-Products, San Gabriel, Calif.). For y-irradiation, microcultures of infected and uninfectedMEF were exposed to a 6'Co source and received atotal of 2,000 rads. Under these conditions, UV irra-diation inactivated 10` PFU of MCMV per ml in 1min, and y-irradiation had no appreciable effect onvirus titer.

Infection ofMEF cultures. Flat-bottomed 96-wellMEF microcultures were infected for stimulation ofthe proliferative response against MCMV and for as-say of cytotoxic activity against MCMV-infected cells.The cultures were decanted, and 1i07 PFU of MCMVin 0.05 ml was inoculated in each well. Infection ofinoculated plates was enhanced by centrifugation at1,900 x g for 20 min (29). Then the culture was allowedto adsorb further for 1.5 h at 370C before addition of0.2 ml ofmedium. After 18 h ofincubation, the cultureswere UV or y-irradiated when indicated, and re-sponder lymphocytes or lymphocytes to be assayedfor cytotoxicity were then added.

Proliferation tests. Flat-bottomed 96-well micro-cultures of MEF were infected with MCMV as de-scribed. Each of three to six replicate wells received106 lymphocytes to be tested in 0.15 ml of lymphocytemedium. After incubation for 48 h at 370C, 0.05 ml ofmedium containing 1.0 iCi of [methyl-3H]thymidine(48 Ci/mmol; Radiochemical Centre, Amersham, Eng-land) was added. After 18 h of incubation at 370C,suspended cells were collected on filter paper with aMASH II multiple automated sample harvester (Mi-crobiological Associates, Bethesda, Md.), washed with10 washes of deionized water, and dried in an oven.The dried sample on filter paper was placed in 5 ml ofscintillation fluid [5 g of 2,5-diphenyloxazole (PPO)and 0.3 g of 1,4-bis-(5-phenyloxazolyl)benzene (PO-POP) in 1.0 liter of toluene), and 3H incorporated incells was counted in a liquid scintillation counter (Tri-Carb, Packard Instrument Co., Downers Grove, Ill.).Secondary in vitro stimulation of lympho-

cytes. To stimulate cytotoxic cells against MCMV(14), MEF monolayers in 24-well plates were inocu-lated with 1060 PFU of TCV in 0.1 and adsorbed for 2h at 370C. After 48 h of incubation, the plates wereUV irradiated as described. Each well then received 5x 10' memory spleen lymphocytes as responders. After5 days of incubation at 370C under 10% C02, viablecells were collected by centrifugation through Iso-paque-Ficoll (9), counted by trypan blue exclusion,and washed before assay or passive transfer.To stimulate cytotoxic cells against ectromelia vi-

rus, the method of Pang and Blanden (30) was fol-lowed. Briefly, stimulators consisted of normal synge-neic spleen cells infected with virulent ectromelia virusat 1 PFU/cell in a volume of 0.3 to 0.4 ml containing107 to 108 cells per ml. Then 8 x 107 responder memoryspleen cells and 8 x 106 stimulator cells were incubatedtogether in 30 ml of lymphocyte medium in a 250-mi,75-cm2 Falcon flask at 390C for 5 days. Viable cellswere collected as above.Assay of cytotoxicity. To assay cytotoxic activity

against MCMV (14), MEF in 96-well flat-bottomed

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plates were infected as described above by centrifu-gation 18 h before the assay and then labeled on theday of assay with 1 to 2 1Ci of 5"Cr (as chromate, 10Ci/ml; National Institute for Radio-Elements, Fleurs,Belgium) per well by incubation for 1 h at 370C. Afterwashing, effector cells were added at calculated effec-tor-to-target (E:T) ratios. Incubation time was 18 h.

For assay of cytotoxicity against ectromelia-infectedcells, the method of Gardner et al. (11) was followed.5"Cr-labeled P-815 cells were incubated with the viru-lent strain of ectromelia virus at a multiplicity of 10for 1 h. After washing, 2 x 103 labeled infected targetswere dispensed per well in 96-well round-bottomedplates (Linbro). Effector cells were added and incu-bated for 6 h.

In both assays, dilutions of effector cells were addedto triplicates of both uninfected and infected targets.After incubation, supernatants were aspirated andcounted in a Packard Auto-Gamma 5210 counter. Thefollowing values were calculated for each E:T ratio:

% lysis of infected or uninfected targets = (% 61Crreleased in presence of effector cells - % 61Crreleased inmedium) x 100/(% of 51Cr in labeledtargets released by water)

% specific lysis = % lysis of infected cells - % lysisof uninfected cells

For each assay, the percentage of specific lysis istandard error was computed and used to expresscytotoxicity. Difference from no specific lysis was cal-culated by the Student t test, and P < 0.05 wasconsidered significant.Treatment with anti-U serum. Approximately 107

lymphocytes in 0.6 ml were incubated for 30 min atroom temperature with 1:6 dilution of anti-U serumpreviously shown to be effective (14). After washing,the cells were incubated with 1:5 guinea pig comple-ment extensively preabsorbed with mouse spleen cells.Cells were washed before assay.Spleen virus titers and passive protection test

Five days after an ip. injection of 10" PFU ofMCMV,the spleen was collected and ground in sterile sand. Itwas then suspended in 1.0 ml of medium and centri-fuged, and 0.05 ml was titrated in duplicate or tripletdecimal dilutions by plaque formation in 24-well MEFplates under an overlay of 0.5% carboxymethyl-cellu-lose in medium. After 4 to 6 days of incubation, theplates were stained with gentian violet and plaqueswere counted under the dissecting microscope. A meantiter was obtained from two or more replicate speci-mens and expressed in logo PFU per spleen ± stan-dard error.To measure the protective effect, cells to be pas-

sively transferred were first centrifuged through Iso-paque-Ficoll (9) and washed. At least two mice re-ceived a designated number of cells i.v. 24 h afterinfection. Four days later, the spleens were collectedfor titration of virus. Protection was expressed as thedifference ofmean log spleen titer in transused animalfrom the mean log spleen titer in untreated controls.The standard error of the difference was computed,and significant protection was measured by the one-tailed Student t test (P < 0.05).Whether protection, as defined here, is correlated

with protection against other measures of morbidityor against mortality was not investigated.

RESULTSAt the onset, attempts to produce a primary

CTL response to MCMV in mice according tothe method of Quinnan et al. (34) yielded vari-able results. In 32 attempts using spleen cellsfrom BALB/c mice of varying ages infected for4 to 19 days, there were 6 successes and 26failures. One thought was that the primary CTLresponse to MCMV may be suppressed by theinfection itself. Three avenues of investigationwere pursued: (i) a method for in vitro secondarygeneration of CTL against MCMV was devel-oped; (ii) the role of passive protection by cellsfrom immune mice and secondary CTL gener-ated in vitro was ascertained; and (iii) the effectof MCMV infection on the primary CTL re-sponse against another virus, ectromelia, wasinvestigated.

Proliferative response. The proliferativeresponse to MCMV was investigated to deter-mine what type of antigenic presentation mightbe suitable for stimulation of CTL in vitro, andto attempt to distinguish the effect of the virusas an antigen and as suppressant of lymphcoyteproliferation (16, 17, 20).Table 1 presents relevant data. In the first

panel, lymphocytes were incubated for 3 dayswith either SGV or TCV MCMV. The presenceof MCMV during incubation directly inhibitedthymidine incorporation by lymphocytes. At106-7 PFU, SGV depressed incorporation morethan TCV (P < 0.01).

In panel 2, immune lymphocytes from mem-ory mice were substituted for lymphocytes fromnormal mice. The results were similar except forcells exposed to 105 7 TCV. These cells incorpo-rated significantly more 3H than did the control,suggesting a specific response.

In the remaining panels, the stated amount ofvirus was adsorbed on MEF cells before beingdiluted. Inoculated cultures were incubated for18 h, and then UV or y-irradiated or left un-treated before addition of lymphocytes. Infec-tion without irradiation produced a small butsignificantproliferative response (7,751 versus3,256 cpm). UV-irradiated cells evoked the bestresponse (15,709 cpm); y-irradiated cells evokeda lesser response (10,034 cpm), and the differ-ence was significant.

In the last three panels, UV-irradiated, in-fected BALB/c (H-2d) fibroblast stimulatorswere exposed to immune CBA (H-2k), A.TL (K5,I}, Dd), or SJL (H-2) lymphocytes from animalswith memory for MCMV. Irrespective ofwhether the stimulator and responder cells

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TABLE 1. Determinants ofproliferative responseaStimulation system Responder lymphocytes [3H]thymidine uptake

MEF MCMV Log Treatment Strain Immuse cpm SE' % of ControlPFU status' p E %o otoNone None 0 None BALB/c NI 8,751 ± 558 ControlNone SGV 5.7 None BALB/c NI 1,735 103 20 (S)dNone TCV 6.7 None BALB/c NI 578 2 7 (S)None TCV 5.7 None BALB/c NI 7,828 ± 361 89 (NS)e

None None 0 None BALB/c I 4,603 ± 142 ControlNone SGV 5.7 None BALB/c I 1,428 ± 327 31 (S)None TCV 6.7 None BALB/c I 1,109± 54 24 (S)None TCV 5.7 None BALB/c I 10,643 ± 772 231 (S)

BALB/c None 0 UV BALB/c I 3,256 ± 402 ControlBALB/c SGV 5.7 None BALB/c I 7,751 ± 322 238 (S)BALB/c SGV 5.7 UV BALB/c I 15,709 ± 839 482 (S)BALB/c SGV 5.7 y BALB/c I 10,034 ± 352 308 (S)

BALB/c None 0 UV CBA I 11,478 ± 1,454 ControlBALB/c SGV 5.7 UV CBA I 33,945 ± 2,854 296 (S)

BALB/c None 0 UV A.TL I 2,773 ± 285 ControlBALB/c SGV 5.7 UV A.TL I 32,376 ± 1,454 1,168 (S)

BALB/c None 0 UV SJL I 9,955 ± 547 ControlBALB/c SGV 5.7 UV SJL I 60,303 ± 2,109 606 (S)a For this experiment, 96 well plates whose wells contained no MEF or 2 x 104 MEF were used. Each datum

represents the mean counts from triplicate wells. In panels 1 and 2, where no MEF were used, 50 pl of SGV orTCV diluted to provide the stated number of PFU was mixed with 106 lymphocytes in a final volume of 200 1d.In the remaining panels, fibroblast well cultures were infected by centrifugation adsorption as described inMaterials and Methods. The virus was washed off after 18 h of incubation and UV or y-irradiated as stated, and106 immune lymphocytes were added per well. All cultures were incubated for 48 h at 37°C before addition of1 jiCi of [3H]thymidine in 0.05 ml per well. Suspended cells were collected 24 h later for counting of 3H. Controlsconsisting of fibroblasts which were UV or y-irradiated with no virus or lymphocytes added did not incorporatemore than 300 cpm (data not shown). Each panel has its own control with which other values are compared.

b NI, Nonimmune lymphocytes from normal BALB/c mice; I, immune lymphocytes from memory miceinoculated 5 weeks previously.

SE, Standard error.S, Significant difference from control by Student's t test (P < 0.05).

'NS, No significant difference.

shared one H-2 antigen, as in the case of A.TL,or none at all, as in the case of CBA and SJLcells, proliferation was stimulated by virus-in-fected cells when lymphocytes from animalswith infected and uninfected MEF were com-

pared.It is clear from the above that infective SGV

and TCV suppressed deoxyribonucleic acid syn-thesis in lymphocytes despite the fact thatMCMV infects these cells very inefficiently (17).However, despite such inhibition, TCV stimu-lated a proliferative response in spleen cells fromimmune animals. Howard et al. (15) had earliershown that inactivated virus could provide thestimulus. The reason why incubation with thesame amount of SGV as TCV produced moreinhibition of lymphocyte deoxyribonucleic acidsynthesis may be that a 1:10 dilution of the

salivary gland stock has a toxic effect, althoughthis type of virus was found to inhibit the mito-genic response of lymphocytes to concanavalinA more than attenuated TCV material (35).

Kelsey et al. (20) first showed that UV-irra-diated infected fibroblasts stimulated the prolif-erative response. Why UV-irradiated ratherthan y-irradiated cells provide the better prolif-erative response is not entirely clear. UV irra-diation may have inactivated all infective virusor an undefined lymphocyte suppressive factor,and y-irradiation did not. Although UV-irradi-ated MCMV has also been reported to suppresslymphocytes (17), cells UV irradiated to thisdegree have reduced macromolecular synthesis(22), whereas -y-irradiated cells can still synthe-size virus and other macromolecules (23).The data also show that there was no apparent

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H-2 restriction of the proliferative response.However, since there are some shared Ia speci-ficities between BALB/c and CBA/H (specific-ities 7 and 15) and between BALB/c and SJL(w30, 20), a requirement for shared Ia antigen inT lymphocytes responding to an infected cell isnot ruled out (32). Another possibility which hasnot been ruled out is a type of "cross-priming"(3), in which the viral antigen may be repro-cessed by macrophages in the responder lym-phocytes. Such a mechanism may also be oper-ative in cases where free MCMV, infective or

inactivated, stimulates a proliferative response.Development of cytotoxic cells against

MCMV. Since UV-irradiated infected fibro-blasts were the best stimulators of the prolifer-ative response against MCMV, a comparison ofdifferent stimulators in developing cytotoxiccells in vitro was made (Table 2). Spleen cellswere obtained from BALB/c mice infected withMCMV 6 weeks previously. As in the case of theproliferative studies, MEF were infected 18 hbefore being UV or y-irradiated. After additionof responders, the cultures were incubated for 5days. Cells generated were assayed for cytotox-icity against MCMV-infected MEF targets (seeMaterials and Methods). As shown in the table,untreated infected MEF were not effective stim-ulators. Both UV and y-irradiated, infectedMEF stimulated specific cytotoxicity, but as inthe case of the proliferative response, UV irra-diation was better. The difference between UVand y-irradiated groups at E:T of 10 was signif-icant (P < 0.05). In a parallel assay when targetswere UV irradiated at 2 h rather than 18 h afterinfection, the 2-h infected targets did not expresscytotoxicity.

In further experiments, UV-irradiated stimu-lators were consistently reliable in stimulatingsecondary cytotoxic lymphocytes in vitro (14).In 39 attempts, we had 36 successes. The cells

generated had no specific cytotoxic activity ifthey were treated with anti-O serum or with anti-Ly 2.2 serum and complement. They only rec-ognized targets which shared the H-2K or Dantigen, or both, and hence possessed the prop-

erties of CTL which lyse virus-infected targets(5).Passive protection by CTL developed in

vitro and H-2 restriction. The direct effect ofCTL developed in vitro was tested in passiveprotection experiments (Table 3). Cytotoxic cellswere generated in vitro in 24-well plates, usingresponder spleen cells from memory mice stim-ulated by UV-irradiated, MCMV-infected MEF(see Materials and Methods). Stimulator andresponder cells were syngeneic. As shown in thetable, the lymphocytes generated in each casehad significant cytotoxic activity when assayedagainst infected MEF. Donor cells ofA.TL originwere protective in syngeneic recipients when10" were given. In the third panel, 1062 cells ofBALB/c origin were protective in BALB/c re-

cipients. In the second panel, cytotoxic cells ofBALB/c origin protected A.TL mice. The twostrains share the same H-2D antigen. On theother hand, cytotoxic cells from CBA mice didnot significantly reduce spleen virus titers ofBALB/c recipients (panel 4). These two strainsdo not share any H-2 antigens. Finally, treat-ment of cytotoxic cells with anti-G serum andcomplement significantly reduced their cyto-toxic activity and their passive protection effectas well (panel 4).Passive protection by lymphocytes from

infected animals. Although CTL generated invitro passively protected infected animals byreducing MCMV titers in the spleen, it is notclear what relationship there is between passiveprotection provided by immune T cells gener-ated in vivo (36) and CTL. If CTL were respon-sible for protection, and they are present in the

TABLE 2. Effect of treatment of stimulating cells on development in vitro of cytotoxic cells against MCMVtargets

Treatment of % Specific lysis of MEF infected with MCMVMCMV MEFsimulators ~ b2o 10 5 2.5 1.3

None . 0 5.3c -1 ± 2.2c 4 ± 1.5c 2 ± 1.8c 2 ± 0.7cUV .59 4.9d 34 ± 4.2d 20 ± 2.1" 13 ± 2.2d 4 ± 1.6dy.ND. 9±..3.1 14± 2.0" 12 ± 2.0d 5 ± 2.6d

a Spleen cells from BALB/c mice with memory for MCMV were incubated with MCMV-infected MEFtreated in three different ways to generate cytotoxic cells in vitro as described in Materials and Methods. After5 days of incubation, viable cells were counted and assayed on MEF-infected fibroblasts as described inMaterials and Methods. The values in this and subsequent cytotoxic assays represent percentage of specificlysis (lysis of infected cells - lysis of uninfected cells) ± standard error of triplicate counts.

b E:T ratio.c No significant specific lysis.d Significant specific lysis according to Student's t test at P < 0.05.'ND, Not done.

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TABLE 3. Passive protection by cytotoxic lymphocytes generated in vitro and H-2 restrictionaDonor cells Recipient mouse

Difference in log Protec-Anti-@ % Specific Log no. Log PFU/spleen titers from control tion'

Strain + comn- lysis of cell Strain SE SEbplement (E:T = 10)ofcls±S

A.TL No 11 ± 2.0d 6.6 A.TL 3.15 ± 0.21 1.01 ± 0.30 SA.TL No Same 6.3 A.TL 4.53 ± 0.07 -0.37 ± 0.21 NS

BALB/c No 29 ± 4.0d 7.2 A.TL 2.75 ± 0.15 1.27 ± 0.23 S

BALB/c No 26 ± 4.0d 7.0 BALB/c 1.30 ± 0.0 1.84 ± 0.27 SBALB/c No Same 6.2 BALB/c 1.75 ± 0.15 1.39 ± 0.34 S

BALB/c No 24 ± 5.0d 6.9 BALB/c 1.30 ± 0.0 1.49 ± 0.23 SBALB/c Yes 5 ± 4.8e 6.9 BALB/c 2.54 ± 0.24 0.25 ± 0.33 NSCBA No 15 ± 3.2d 6.9 BALB/c 2.75 ± 0.27 0.04 ± 0.24 NS

a Cytotoxic cells of donor strain were first generated in vitro. They were assayed and passively transferred tomice which had received 104 PFU of MCMV ip. 24 h previously. Four days later, spleens were collected forvirus titration. Each titer represents the geometric mean of at least two assays. Each panel had a set of at leasttwo control mice which received no cells. Protection was measured by difference of observed spleen titers fromcontrol titers. Standard error of difference was computed from standard errors of the two titers. Protection wasmeasured by the one-tailed Student t test at P < 0.05.

b SE, Standard error.CS Significant protection; NS, no significant protection.dSignificant specific lysis.'No significant specific lysis.

spleen only for a short time after infection (34),then one might expect a relatively short periodof time after infection when spleen cells couldprovide passive protection. To test this point,spleen cells from mice infected 1 to 30 dayspreviously were passively transferred to recipi-ents which had received 104.0 PFU ofMCMV i.p.24 h previously. Four days later, the spleenswere titered for their virus content (Table 4). Itis clear that 107 cells from mice infected 1 and 3days previously were not protective. This num-ber was protective when cells came from miceinfected 6, 10, or 14 days previously. There is asuggestion that 1075 cells from a mouse infected30 days previously were protective, althoughsignificance was not attained. The accentuationof infection by cells from 3-day-infected animalsis unexplained. It may be related to the immu-nosuppressive effect of infected spleen cells atthis stage of infection. Virus titers are at theirpeak around 3 days after infection (27). Trans-ferred cells may also have increased the virusload without having increased the pool of im-mune cells.H-2 restriction of passively transferred

cells. Since CTL against MCMV-infected cells,like CTL in other virus infections, are H-2D orH-2K restricted in their recognition of targets(34, 40, 14), it would appear that if they wereresponsible for the passive protection describedabove, such protection should also be H-2 re-stricted. To test this point, animals from various

TABLE 4. Protective effect of spleen cells from miceinfected from 1 to 30 days'

Donor Log no. Recipient's log Difference inmice in- of cells PFU/spleen ± log titer from *ohfected trans- SE control ± SE tiondays ago ferret

1 7.0 4.51 ± 0.06 0.06 ± 0.31 NS3 7.0 5.39 ± 0.06 -0.82 ± 0.31' NSd6 7.0 2.97 ± 0.19 1.60 ± 0.36 S

6.5 3.74 ± 0.01 0.83 ± 0.30 S5.5 5.25 ± 0.13 -0.68 ± 0.33 NS

10 7.0 3.25 ± 0.17 1.32 ± 0.34 S14 7.0 2.95 ± 0.05 1.62 ± 0.30 S30 7.5 3.80 ± 0.70 0.77 ± 0.76 NS

7.0 4.81 ± 0.39 -0.24 ± 0.49 NS

Donor and recipient A.TL mice received 104 PFUofMCMV i.p. as described in Materials and Methods.Each value represents the mean of at least two recip-ients. Virus titers are expressed in log PFU per spleen± standard error (SE). The control titer from eightanimals which received no cells was 4.57 ± 0.30. Pro-tection was measured by the one-tailed Student t test(P < 0.05).

b NS, No significant protection; S, significant pro-tection.

Negative values indicate accentuation of infection.dSignificant for accentuation of virus titer (P <

0.05).

donor strains were infected with 104 PFU ofMCMV 6 days before their spleen cells weretransferred i.v. to A.TL and BALB/c recipientswhich were infected 1 day before (Table 5).Spleen virus was titrated 4 days later. Immune

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TABLE 5. H-2 restriction ofprotective effect of spleen cells from 6-day-infected micea

Donor Log no. of H-2 antigen Recipient log PFU/ Difference in log titeripientsen train shared spleen ± SEb from control ± SE Protection

A.TL .. 7.0 A.TL AU 2.45 + 0.21 2.54 + 0.26 SBALB/c .. 7.0 A.TL D 3.05 ± 0.15 1.94 ± 0.21 SSJL ... 7.0 A.TL K 3.46 ± 0.17 1.53 ± 0.23 S

CBA. 7.3 A.TL I 4.47 ± 0.13 -0.77 ± 0.16 NSdC57BL .. 7.3 A.TL None 4.31 ± 0.14 -0.61 ± 0.17 NS

BALB/c 7.0 BALB/c Al 2.39 ± 0.61 1.78 ± 0.61 SA.TL.7.0 BALB/c D 2.40 ± 0.10 1.77 ± 0.12 S

a Mean control log virus titer for recipient A.TL mice was 4.99 ± 0.15 in first three rows and 3.70 ± 0.09 forsecond two rows. The control log virus titer for BALB/c recipients in the last two rows was 4.17 ± 0.07. AUdonor animals were infected 6 days before cell transfer. Control values were obtained from titration of threereplicates, and recipient virus titers represent a minimum of two.

b SE, Standard error.cS. Significant for protection; NS, not significant (see footnote a, Table 3).d Significant accentuation of virus titer (P < 0.05).

spleen cells from C57BL mice did not protectA.TL mice, these two strains not sharing any H-2 antigen. CBA cells did not protect A.TL micedespite the fact that they shared I antigens. Onthe other hand, in the case of A.TL and BALB/c strains, their cells protected homologous recip-ients as well as heterologous recipients. Thesetwo strains share the same H-2Dd antigen. Thus,it appears that protection by spleen cells from 6-day-infected mice is H-2K or H-2D restricted,and that the protection is probably mediated byCTL, although we could not consistently dem-onstrate them by in vitro assays.We have shown that secondary CTL can be

generated in vitro, that they are protective inthe animal, and that primary CTL may also bedemonstrated by the passive protection of in-fected animals. This is indirect evidence that theprimary CTL response exists as reported byQuinnan et al. (34). Whether the response is asstrong as in the case of other virus infections isdifficult to assess. In addition to our difficulty indetecting primary CTL by in vitro assays, onemight also note that the amount of specific lysisreported by Quinnan et al. (34) was relativelylow and the E:T ratio at which they demon-strated lysis was quite high (100:1). We ap-proached the problem ofwhetherMCMV affectsthe CTL response by studying the effect ofMCMV infection on the well-described primaryCTL response against ectromelia virus (5).Effect ofMCMV on primary cytotoxic re-

sponse against ectromelia virus. Three typesof experiments were done. In the first, groups ofmice were infected with 104 PFU of MCMV(Table 6). Three, 7, and 14 days later, they werealso infected with 2 x 104 PFU of attenuatedectromelia virus to stimulate their cytotoxic cells

TABLE 6. Effect ofMCMV infection on primarycytotoxic response ofmice against ectromelia virus'

MCMV injected % Specific lysis of spleen cells againstectromelia infected targetsdays before ec -_______________tromelia 64b 32 16

NoMCMV 20 ± 2.8 14 ± 2.3 9 ± 1.73 3 ± 1.3c 0.5 ± 0.3 2 ± 1.7c4 2 ± 0.7c 2 ± 0.5c 1 ± 0.314 18± 5.Od 13± 3.7d 7 ± 3.3d

a Control values (no MCMV) represent the meanand standard errors of quadruplicate separate assays,each representing cells from a separate mouse, andthe other values represent duplicate assays.b E:T ratio.

c Significant difference from control cytotoxic assaywithout MCMV infection at corresponding E:T (firstrow) according to Student's t test (P < 0.05).

d No significant difference.

against this virus. Five days later, the spleenswere collected and lymphocytes were assayed onectromelia-infected P-815 targets as described inMaterials and Methods. MCMV administered 3or 7 days before ectromelia virus significantlysuppressed the development of cytotoxic cellsagainst ectromelia-infected targets (Table 6).However, MCMV given 14 days before ectro-melia virus had no such effect.

In the second type of experiment, the effect ofacute MCMV infection of mice with memory forectromelia virus on the ability of spleen cellsfrom such mice to be stimulated in vitro wastested. MCMV was injected 3 days before spleencells were collected from memory mice. Thesecells were then stimulated by ectromelia-in-fected spleen cells by 5 days of incubation invitro as described in Materials and Methods. At

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E:T ratios varying from 64 to 4, there was sig-nificant reduction of cytotoxic activity againstectromelia-infected targets (Table 7).

In a third series of experiments, two BALB/cmice were first infected with 104 PFU ofMCMVi.p. 3 days before an i.v. injection of 2 x 104 PFUofattentuated ectromelia virus. Five weeks later,spleen cells were collected from these mice andfrom two control mice with memory for ectro-melia virus but which did not receive MCMV.These two sets of cells were stimulated in vitroin three flask cultures to generate secondaryCTL: one for the control cells and two for cellsfrom MCMV-infected mice. Cytotoxic assay ofcells generated showed that the percentages ofspecific lysis for the control and two test samplesat E:T of 16 were 45 ± 3.0,9 + 0.7,and 12 ± 1.7.The difference between the control and each ofthe test assays was significant (P < 0.05).Thus MCMV infection inhibits (i) the devel-

opment of a primary cytotoxic response; (ii) theability of lymphocytes from mice with estab-lished memory to develop cytotoxic cells; and(iii) the ability of mice to develop memory cellsfor cytotoxic response to ectromelia virus.

DISCUSSIONThe development of a system to generate sec-

ondary CTL against MCMV in vitro has per-mitted us to study the conditions of generation,properties of cells generated, and their effect oninfected animals (14). We show here that theyreduced spleen MCMV virus titers when as littleas 1.6 x 106 cells are passibly transferred (Table3). Protection was H-2 restricted and abrogatedby anti-@ serum. These experiments show thatcell preparations containing specific CTL againstMCMV infected targets can protect.That they are present and do protect during

acute infection is suggested by the following. (i)Spleen cells were most protective if they camefrom animals which had been infected 6 to 14

days previously. Cells from animals infected ear-lier or later were less protective. (ii) Protectionby spleen cells from animals infected for 6 dayswas also H-2 restricted.

In support of the above interpretation, Starrand Allison (36) had already shown that passiveprotection against death imparted by immunecells from mice infected for 6 days was destroyedby treatment with anti-@ serum and comple-ment. Quinnan et al. (34) demonstrated by invitro assays that primary CTL were present inspleens of mice 3 to 20 days after infection, witha peak around 10 days.

Final proof that CTL have the assumed func-tions in infected animals may require (i) the invitro generation of pure specific CTL and dem-onstration that they protect and (ii) demonstra-tion that virus-infected cells are actually lysedin vivo. Methods to test these theses remain tobe developed.

It should be pointed out that the assay methodfor passive protection used in these studies isquantitatively inadequate to exclude minor pro-tective or even infection accentuation rolesplayed by other factors. It was occasionallynoted that when immune allogeneic lympho-cytes were injected, the spleen virus titers wereat times increased (Table 5). Whether this is achance phenomenon or whether it is related toaccentuation of spleen titers of MCMV by allo-geneic reaction of either donor versus host or

host versus donor (10, 38) is unknown. By thesame token, the assay method for cytotoxicitydoes not permit us to say with confidencewhether the cytotoxic response against MCMVdeveloped in vivo or in vitro is quantitativelycomparable with that of other virus systems.Our experience is that the primary in vivo re-

sponse cannot always be deleted, but CTL gen-erated in vitro almost always can. The assaymethods and targets for assaying both types ofCTL were identical.

TABLE 7. Effect ofMCMV infection on cytotoxic lymphocytes generated in vitro from memory cellsaMemory % Specific lysis of cultured cells against ectromelia infected targetsmice in-fectedwith 64b 32 16 8 4 2MCMV

No ......... 60 + 8.0 47 ± 6.0 34 ± 11 21 ± 7.8 11 ± 5.5 6± 3.5Yes ........ 21±9.0c 13±7.0c 6±2.7c 2±1.6c 1 ±0.9c 1±O0.3d

a Memory mice were inoculated with ectromelia 6 weeks before collection of spleen cells. In addition, somemice were inoculated with MCMV 3 days before collection. Spleen cells were stimulated in vitro with ectromeliavirus-infected spleen cells for 5 days at 390C. Cultured cells were then assayed for cytotoxicity. The controlvalues (no MCMV) represent assays of cells from duplicate cultures, and the second row represents quadruplicatecultures each from a separate animal.

b E:T ratio.c Significant reduction by t test from control specific lysis (no MCMV) at designated E:T (P < 0.05).d No significant difference.

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T CELLS IN CYTOMEGALOVIRUS INFECTION 775

The early stimulation of CTL after viral infec-tions is thought to play an important role in thelysis of virus-infected cells and in the recoveryfrom infection (4; Zinkernagel and Doherty, in

press). Since this mechanism exists after cyto-megalovirus infection and seems to operate inacute infection, the question may still be askedwhether it is either qualitatively or quantita-tively deficient. Specifically, why are specificCTL unable to eliminate completely all virus-infected cells and prevent the persistence ofinfection? In this regard, several peculiarities ofherpes simplex virus and cytomegalovirus maybe considered.

First, Pfizenmaier et al. (33) reported thatmice acutely infected with herpes simplex didnot develop specific CTL in vivo. Only by isolat-ing and incubating sensitized lymphocytes fromlymph nodes draining a site of infection did suchcells develop. Cyclophosphamide treatment ofthe animals also facilitated a CTL response. Thehypothesis was that a suppressor mechanism forCTL development operating in vivo had to becircumvented. Whether such a mechanism op-erates in the case of MCMV is not known. Pos-sibly, it was circumvented during secondary invitro stimulation.

Second, evidence has been presented thatMCMV infection suppressed the developmentof the primary CTL response against ectromeliavirus. It also inhibited cells from mice with es-tablished memory for ectromelia to differentiateinto cytotoxic cells. Finally, it suppressed theexpansion of memory cells for the cytotoxic re-sponse. These findings are in line with otheroverwhelming evidence that cytomegalovirus in-fections of humans and mice are immunosup-pressive (8, 27), although we have not shownthat these findings are unique for MCMV. Dur-ing acute infection in mice, particularly within 1to 2 weeks, antibody formation against sheepleukocytes, interferon production, response tospecific and nonspecific mitogens, and skin graftrejection are all suppressed (16, 27, 28). Kelseyet al. (20) suggested that such immunosuppres-sion did not extend to MCMV itself, since theymeasured a specific proliferative response. Os-born et al. (27), however, suggested that theimmunosuppressive nature ofMCMV accountedfor the late development of antibodies. WhetherMCMV infection suppresses homologous im-munity is still a moot question. To answer it,one would ideally compare immune responses inthe presence and absence of active infection.This may be particularly difficult in the case ofdevelopment of CTL, since this response may bedifficult to elicit in the absence ofactive infection(Zinkernagel and Doherty, in press).A piece of indirect evidence that specific ho-

mologous immunity may be suppressed is thatfibroblasts actively infected with MCMV are apoor antigen for both the proliferative and cy-totoxic responses. Only after infected cells hadbeen treated by UV irradiation, which presum-ably eliminates the lymphocyte-suppressive na-ture of either the virus or a cell-virus product,was the cell-associated virus rendered optimallyimmunogenic. Further, we have been unable touse as in vitro stimulators of the cytotoxic re-sponse syngeneic macrophages or lymphocytesadsorbed with MCMV (14). Macrophages arevery inefficiently infected by MCMV, particu-larly SGV (24). These are the cells which, wheninfected with other viruses, are most effective instimulating a CTL response (32). Hence, in acuteinfections of animals with MCMV, optimal cell-associated antigens for the stimulation of thecytotoxic response may not be available.

Finally, the recognition of MCMV-infectedcells by specific cytotoxic cells may also be de-fective compared with other virus systems. It isnow assumed that in order for cytotoxic lympho-cytes to recognize a virus-infected cell, virusantigens must be expressed on the cell surface.This expression can occur quickly and early inthe replicative cycle (1). Even in the case ofherpes simplex virus, target cells were recog-nized by cytotoxic lymphocytes 30 to 90 minafter infection (33). In the case ofMCMV, fibro-blast targets were not recognized after 2 h ofinfection, and targets were routinely infected for18 h before assay for cytotoxicity (14, 34). BothB and T lymphocytes have been implicated inlatent or persistent infection (26, 39). It is pos-sible that in the MCMV system, cytotoxic cellsdo not recognize early infected cells, or they maynot recognize certain genres of cell-virus associ-ations. Their efficiency in comparison with othervirus systems may be questioned.Thus, further investigation should better

characterize the stimulus and targets of the CTLgenerated in CMV infections. The role of CTLin surveillance against infected cells in chronicviral infections and the immunological condi-tions which permit infected cells to persist orproliferate remain to be elucidated.

ACKNOWLEDGMENTSThe author was faculty scholar, Josiah Macy, Jr. Founda-

tion, while doing this work. This study was also supported bythe Australian National University and Public Health Servicegrant 5 ROI-AI-11798 from the National Institutes of Health.I also thank G. L. Ada for the facilities of his department andR. B. Ashman for his generous help. They and R. V. Blandenalso provided stimulating discussions and criticisms.

LITERATURE CITED1. Ada, G. L., D. C. Jackson, R. V. Blanden, R. Tha lla,

and N. A. Bowern. 1976. Changes in the surface ofvirus-infected cells recognized by cytotoxic T cells. I.

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Minimal requirements for lysis of ectromelia-infectedP-815 cells Scand. J. Immunol. 5:23-30.

2. Araullo-Cruz, T. P., M. Ho, and J. A. Armstrong.1978. Protective effect of early serum from mice aftercytomegalovirus infection. Infect. Immun. 21:840-842.

3. Bevan, ML J. 1976. Cross-priming for a secondary cyto-toxic response to minor H antigens with H-2 congeniccells which do not cross in the cytotoxic assay. J. Exp.Med. 143:1283-1288.

4. Blanden, R. V. 1974. T cells response to viral and bacte-rial infection. Transplant. Rev. 19:56-88.

5. Blanden, R. V. 1977. Cell-mediated immune response toacute viral infection. Prog. Immunol. 1:463-471.

6. Blanden, R. V., N. A. Bowern, T. E. Pang, I. D.Gardner, and C. R. Parish. 1975. Effects of thymus-independent (B) cells and the H-2 gene complex onantiviral function of immune thymus-derived (T) cells.Aust. J. Exp. Biol. Med. Sci. 53:187-195.

7. Blanden, R. V., and I. D. Gardner. 1976. The cell-mediated immune response to ectromelia virus infec-tion. I. Kinetics and characteristics of the primary ef-fector T cell response in vivo. Cell. Immunol. 22:271-282.

8. Chatterjee, S. N., M. Fiala, J. Weiner, J. A. Stewart,B. Stacey, and N. Warner. 1978. Primary cytomega-lovirus and opportunistic infections. J. Am. Med. Assoc.240:2446-2449.

9. Davidson, W. F., and C. R. Parish. 1975. A procedurefor removing red cells and dead cells from lymphoid cellsuspensions. J. Immunol. Methods 7:291-300.

10. Dowling, J. N., B. C. Wu, J. A. Armstrong, and M.Ho. 1977. Enhancement of murine cytomegalovirus in-fection during graft versus host reaction. J. Infect. Dis.135:990-994.

11. Gardner, I., N. A. Bowern, and R. V. Blanden. 1974.Cell-mediated cytotoxicity against ectromelia-infectedtarget cell. Eur. J. Immunol. 4:63-67.

12. Ho, M. 1977. Virus infections after transplantation in man.Arch. Virol. 55:1-24.

13. Ho, M. 1978. Cytomegalovirus infections and diseases.Dis. Mon. 24:1-61.

14. Ho, M., and R. B. Ashman. 1979. Development in vitroof cytotoxic lymphocytes against murine cytomegalo-virus. Aust. J. Exp. Biol. Med. Sci. 57:425-428.

15. Howard, R. J., D. M. Mattason, M. V. Seidel, and H.H. Balfour, Jr. 1978. Cell-mediated immunity to mu-rine cytomegalovirus. J. Infect. Dis. 138:597-604.

16. Howard, R. J., J. Miller, and J. S. Najarian. 1974.Cytomegalovirus-induced immune suppression. II. Cell-mediated immunity. Clin. Exp. Immunol. 18:119-126.

17. Hudson, J. B., L. Loh, V. Misra, B. Judd, and J.Suzuki. 1977. Multiple interactions between murinecytomegalovirus and lymphoid cells in vitro. J. Gen.Virol. 38:149-159.

18. Kees, U., and R. V. Blanden. 1976. A single geneticelement in H-2K affects mouse T-cell antiviral functionin poxvirus infection. J. Exp. Med. 143:450455.

19. Kees, U., and R. V. Blanden. 1977. Protective activityof secondary effector T cells generated in vitro againstectromelia virus infection in vivo. Clin. Exp. Immunol.30:338-346.

20. Kelsey, D. K., J. C. Overall, Jr., and L A. Glasgow.1978. Correlation of the suppression of mitogen respon-siveness and the mixed lymphocyte reaction with theproliferative response to viral antigens of splenic lym-phocytes from cytomegalovirus-infected mice. J. Im-munol. 121:464-470.

21. Klein, J., L. Flaherty, J. L. Van de Berg, and D. C.Shreffler. 1978. H-2 haplotypes, genes, regions andantigens: first listing. Immunogenetics 6:489-512.

22. Lafferty, K. J., I. S. Misko, and M. A. Cooler. 1974.

Allogeneic stimulation modulates the in vitro responseof T cells to transplantation antigen. Nature (London)249:275-276.

23. Levine, S. 1963. Effects of X-irradiation on the responseof animal cells to virus. Prog. Med. Virol. 5:127-168.

24. Mims, C. A., and J. Gould. 1978. The role of macro-phages in mice infected with murine cytomegalovirus.J. Gen. Virol. 41:143-153.

25. Naimias, A. J., and B. Roizman. 1973. Infection withherpes simplex virus 1 and 2. N. Engl. J. Med. 289:781-789.

26. Olding, L B., F. C. Jensen, and M. B. A. Oldstone.1975. Pathogenesis of cytomegalovirus infection. I. Ac-tivation of virus from bone-marrow derived lympho-cytes by in vitro allogeneic reaction. J. Exp. Med. 141:561-572.

27. Osborn, J. E., A. A. Blazkovec, and D. L Walker.1968. Immunosuppression during acute murine cyto-megalovirus infection. J. Immunol. 100:835-844.

28. Osborn, J. E., and D. N. Medearis, Jr. 1967. Suppres-sion of interferon and antibody and multiplication ofNewcastle disease virus in cytomegalovirus infectedmice. Proc. Soc. Exp. Biol. Med. 124:347-353.

29. Osborn, J. E., and D. L. Walker. 1968. Enhancement ofinfectivity of murine cytomegalovirus in vitro by cen-trifugal inoculation. J. Virol. 2:853-854.

30. Pang, T., and R. V. Blanden. 1976. The cell-mediatedimmune response to ectromelia virus infection. Second-ary response in vitro: specificity, nature of effector andresponder cells and requirements for induction of anti-genic changes in stimulator cells. Aust. J. Exp. Biol.Med. Sci. 54:253-264.

31. Pang, T., and R. V. Blanden. 1977. Genetic factors inthe stimulation of T cell responses against ectromeliavirus-infected cells: role of H-2K, H-2D and H-21 genes.Aust. J. Exp. Biol. Med. Sci. 55:549-559.

32. Pang, T., and R. V. Blanden. 1977. Requirements forstimulation of T cell responses against virus-infectedcells: nature of ectromelia virus infected cells capable ofstimulating cytotoxic T cells in a secondary response invitro. Aust. J. Exp. Biol. Med. Sci. 55:539-547.

33. Pflzenmaier, K., IL Jung, A. Starzinski-Powitz, M.Rollinghoff, and M. Wagner. 1977. The role ofT cellsin anti-herpes simplex virus immunity. J. Immunol.119:939-944.

34. Quinnan, G. V., J. E. Manischewitz, and F. A. Ennis.1978. Cytotoxic T lymphocyte response to murine cy-tomegalovirus infection. Nature (London) 273:541-543.

35. Selgrade, M. K., A. Ahmed, K. W. Sell, M. E. Gersh-win, and A. D. Steinberg. 1976. Effect of murinecytomegalovirus on the in vitro response of T and Bcells to mitogens. J. Immunol. 116:1459-1465.

36. Starr, S. E., and A. C. Allison. 1977. Role ofT lympho-cytes in recovery from murine cytomegalovirus infec-tion. Infect. Immun. 17:458-462.

37. Yap, K. L, G. L Ada, and I. F. C. McKenzie. 1978.Transfer of specific cytotoxic T lymphocytes protectsmice inoculated with influenza virus. Nature (London)273:238-239.

38. Wu, B. C., J. N. Dowling, J. A. Armstrong, and M.Ho. 1975. Enhancement of mouse cytomegalovirus in-fection during host versus graft reaction. Science 190:56-58.

39. Wu, B. C., and M. Ho. 1979. The characteristics ofinfection of B and T lymphocytes from mice afterinoculation with cytomegalovirus. Infect. Immun. 24:856-864.

40. Zinkernagel, R. M., and P. C. Doherty. 1974. Restric-tion of in vitro T cell-mediated cytotoxicity in lympho-cytic choriomeningitis with a syngeneic or semialloge-neic system. Nature (London) 248:701-702.

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