INFECTION AND IMMUNITY, Apr. 1975, p. 622-629Copyright 0 1975 American Society for Microbiology

Vol. 11, No. 2Printed in U.S.A.

T Lymphocyte Function as the Principal Target ofLymphocytic Choriomeningitis Virus-Induced

ImmunosuppressionK. BRO-J0RGENSEN,* F. GOTTLER, P. N. JORGENSEN, AND M. VOLKERT

Institute of Medical Microbiology, University of Copenhagen, Copenhagen, Denmark

Received for publication 14 November 1974

Plaque-forming cell responses against sheep erythrocytes, Escherichia colilipopolysaccharide, pneumococcal polysaccharide, and polyvinylpyrrolidonewere examined in mice infected with lymphocytic choriomeningitis virus. A 92 to96% reduction of the thymus-dependent anti-sheep erythrocyte responses wasobserved 2 to 4 weeks after infection. However, the thymus-independent re-sponses against the three other antigens were close to normal at all stages of theinfection. Studies on allograft immunity of infected C3H mice against DBA/2mastocytoma cells revealed a severe suppression of the T cell-mediated cytotoxicresponse which was temporally related to the impaired humoral responsivenessagainst sheep erythrocytes. The capacity of spleen cells from infected mice to re-store immune responsiveness of lethally irradiated recipients against sheeperythrocytes was significantly reduced. The adoptive responses, however, wereclearly improved when normal thymus cells were added to the inferior spleencells. Moreover, it appeared that the spleen cells from immunosuppressed donormice could not confer suppression to normal lymphoid cells. The presented find-ings are consistent with the assumption that a numeric deficiency of T cells, orcells belonging to some T cell subpopulation, is the primary cause of lymphocyticchoriomeningitis virus-induced immunosuppression.

The mechanisms by which viruses may de-press the function of the immune apparatushave remained a controversial subject (10, 32,36, 44). In a recent paper (5) on the im-munosuppressive effect of the lymphocytic cho-riomeningitis (LCM) virus it was reported thatapparently this virus acted by causing a numer-ical deficiency of cells belonging to the popula-tion of radiosensitive immunocompetent cells.Moreover, it was suggested that this cellulardeficiency was probably a consequence of anLCM virus-induced suppression of the lymph-oid precursor cells (4, 5). These investigations,however, were not designed to ascertain whetherthe LCM virus infection affected preferentiallybone marrow-derived (B) or thymus-processed(T) lymphocytes, or whether perhaps both thesecell populations were influenced.From an earlier study on the LCM virus (27),

it appears that the virus interferes with humoralimmune responses against thymus-dependentantigens such as sheep erythrocytes (SRBC)and human serum albumin. Moreover, someobservations have been reported which mightsuggest a concomitant suppression of cell-mediated immunity. Thus, the footpad re-sponses of LCM virus-infected mice against

unrelated viruses were reduced (27), and dataon skin allograft survival indicated a signifi-cant, although very moderate, enhancing effectof the virus (24). In the present work, B and Tcell functions in LCM virus-infected animalswere investigated by employing a number ofthymus-independent antigens and by studyingthe allograft responses in infected mice bymeans of the in vitro measurements of cytotoxiccells. The observations indicated a significantsuppression of T cell-dependent immune func-tions exclusively. The findings were supportedby data which were obtained in cell transferexperiments. These data were consistent withthe assumption that a numerical deficiency of Tcells was the primary cause of the LCM virus-induced immunosuppression and, in addition,they argued against mechanisms involving sup-pressor cells or suppressive humoral factorswhich have been reported to play a role inantigenic competition (12, 40, 41) and in immu-nosuppression with other viruses (14, 22).

MATERIALS AND METHODSVirus. The*LCM virus strain employed was the

Traub strain. This strain was obtained from E. Traub(Ttibingen, Germany) in 1960. In this laboratory the

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virus was kept at -70 C as 10% stock suspensions ofspleens from intraperitoneally (i.p.) infected C.Hmice. A total of 20 mouse passages were carried out.The virus preparations used in this study were tissueculture fluid obtained after three or four additionalpassages of the virus in monolayers of L cells grown inEagle minimum essential medium. The preparationswere kept at -70 C. Dilutions of the virus wereprepared in phosphate-buffered saline (PBS), andvirus titrations were carried out as previously de-scribed (5) by intracranial inoculation into whiteSwiss mice.

Mice. Except for those used for titration purposes,all animals employed were strictly inbred C3H/Ssclmice (originally obtained from Statens Seruminstitut,Denmark). The mice were 2 to 3 months old, and intransplantation experiments cell donors and recipi-ents were of the same sex. Infections were produced byi.p. inoculation of 103 mean lethal doses (LD50) ofLCM virus. This injection causes a symptomless andtransient infection with maximum viremia on day 6 to8 (5).

Antigens and immunizations. SRBC stored inAlsever solution were washed three times in PBS. Thesuspension was adjusted to contain about 2 x 108SRBC per 0.5 ml, which was injected i.p. or intrave-nously (i.v.) as described. Lipopolysaccharide (LPS)of Escherichia coli 055:B5 was obtained from DifcoLaboratories, Detroit, and was extracted by themethod of Westphal et al. (43). A solution containing1 mg of LPS per ml of PBS was boiled for 1 himmediately before use. After centrifugation the solu-tion was adjusted to contain 50 ,ug of LPS per 0.5 ml,which was injected i.v. Type III pneumococcal poly-saccharide (S III), made as described by Katz andPappenheimer (20), was obtained from Wellcome,England, and 0.5 1g of S III in 0.5 ml of PBS was usedfor i.v. immunization. Polyvinylpyrrolidone (PVP) K90, from Fluka AG, Switzerland, was dissolved in PBS,and amounts of 0.5 gg in 0.5 ml were given i.v. Theimmunization doses employed were all selected so asto give rise to near-optimal immune responses againstthe respective antigens.P-815-X2 mastocytoma cells of DBA/2 origin were

kindly donated by K. T. Brunner and J.-C. Cerottini,Lausanne, Switzerland. The ascites tumor was main-tained by serial transplantations into the strain oforigin. For immunization, 5 x 106 cells in 0.5 ml ofPBS were injected i.p.

Assays for PFC. Individual lymphoid cell suspen-sions were prepared by passing the spleens throughstainless-steel meshes. After a wash in Eagle mini-mum essential medium with Hanks salts, appropriatedilutions of the spleen cells were assayed for plaque-forming cells (PFC), using SRBC or antigen-coatedSRBC (see below). Agarose A 37 (L'Industrie Bi-ologique Francaise S.A.), 0.5% in Eagle minimumessential medium (0.4 ml), 10% SRBC (0.05 ml), andthe spleen cell suspensions (0.1 ml) were mixed andpoured onto microscope slides precoated with 0.1%agarose in water. The slides were incubated for 1 h at37 C in a humid atmosphere containing 5% CO2.Guinea pig serum diluted 1:10 in Eagle medium wasadded, and the incubation was continued for 2 h. The

plaques were counted by indirect illumination with amagnifying glass.

LPS-coated SRBC were prepared as described byMoller and Michael (30). Packed and washed SRBC(0.1 ml/ml) were added to a boiled solution containing1 mg of LPS per ml of PBS. After incubation at 37 Cfor 30 min, the SRBC were washed three times beforeuse in the PFC assay. S III-coated SRBC were madeas described by Baker et al. (2). Amounts (0.5 ml) ofpacked SRBC were suspended in 1.0 ml of salinecontaining 1 mg of S III. Then 1.0 ml of 0.1%chromium chloride (CrCl3. 6H20) in saline was addedunder mixing. Incubation was carried out at roomtemperature for 5 min and followed by four washeswith saline. The coating with PVP was performedessentially as described by Anderson and Blomgren(1). SRBC pretreated with 1:20,000 tannic acid at37 C for 15 min were washed twice in PBS. To a 4%suspension of the tanned cells was added an equalamount of PBS containing 800 Mg of PVP K 15 (FlukaAG, Switzerland) per ml. After incubation of themixture at room temperature for 15 min, the labeledcells were washed three times with PBS.

Splenic PFC were always calculated from thecounts of duplicate assays of individual spleen cellsuspensions. As to the PFC responses against LPS, SIII, and PVP, the numbers of PFC were corrected forbackground PFC, which were determined by testingthe spleen cells against normal or tannic acid-treatedSRBC. PFC counts of normal nonsensitized mice didnot exceed 200 PFC per spleen in tests carried outagainst any of the four antigens under study.

In vitro measurement of cytotoxic cells. Spleencells suspensions were prepared by homogenizingpooled spleens by one stroke by hand in a Ten-Broekgrinder. After sedimentation for 45 min at 4 C, thecells of the supernatant fluid were washed four timesin Parker medium without serum added by centrifu-gation at 400 x g for 10 min, and finally the lymphoidcells were resuspened in Parker medium with 10%inactiviated fetal calf-serum so as to contain 107 cellsper ml. DBA/2 mastocytoma target cells were grownin suspension culture in Eagle minium essentialmedium with 10% inactiviated fetal calf serum. Forlabeling, the technique described by Cerottini andBrunner (6) was followed by incubating 5 x 10" cellsat 37 C for 30 min in 1 ml of medium containing 50MCi of 5'Cr as Na.CrO4 (Amersham, England, speci-fic activity >200 mCi/mg). After five washes the cellswere resuspended in medium at a concentration of 105cells per ml.

The quantitation of the 5'Cr release from targetcells was performed as previously described (19).Briefly, volumes of 0.6-ml reaction mixtures contain-ing 3 x 104 labeled target cells and 50 or 100 times 3 x104 spleen cells were incubated in test tubes set up inroller drums (20 rotations/h). After imcubation for 18h at 37 C, the tubes were centrifuged for 10 min at1,000 x g, and 0.4 ml of the supernatant was added tothe scintillation fluid containing the nonionic deter-gent BBS-3 (Beckman). Radioactivity was measuredin a Beckman LS-233 liquid scintillation counter. Thetotal and the spontaneous 5'Cr release was deter-mined for each preparation of target cells employed.

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Total release was determined by adding 2 x 104 targetcells to the scintillation fluid, and ranged between19,000 and 21,400 counts/min. Spontaneous releasewas measured in target cell cultures incubated asdescribed but without spleen cells added, and rangedbetween 5,900 and 7,900 counts/min. The cytotoxic-ity of a given spleen cell suspension was expressed asthe mean counts per minute obtained from fiveparallel cultures minus the mean counts per minutespontaneously released. The variation which was

recognized between parallel cultures was always mini-mal and has therefore been omitted from the tables.Lymphoid cell transfer experiments. Cell suspen-

sions were prepared by passing pooled donor spleensor thymuses through stainless-steel meshes. After twowashes with Hanks salt solution, cell counting, ad-justing, and mixing were performed so that thedesired cell transplantation doses were contained involumes of 0.5 ml. Recipient mice were treated with800 R of X rays. The irradiation was administered bya Siemens Stabilipan therapy machine operated withthe following factors: 200 kV, 15 mA, 1.0-mm copperfiltration. The dose rate was 47 Rlmin, and half-valuelayer was 1.5-mm copper. Within 3 h after irradiationthe recipients were injected i.v. with the donor cells,and immediately afterwards they received 2 x 108SRBC i.p.Complement fixation test. Complement-fixing

antibodies against LCM virus antigen were measuredby a technique previously described (3).

RESULTS

PFC responses of LCM virus-infected miceagainst thymus-dependent and thymus-inde-pendent antigens. In the first series of experi-ments, the thymus-dependent immune re-

sponses to SRBC and the thymus-independent

immune responses to LPS (1, 30), S III (17), andPVP (1) were examined at various times postin-fection with LCM virus. In all experiments thesame time schedule was employed. On days 24,14, and 4 before immunization, groups of fivemice were inoculated i.p. with 103 LD50 of thevirus. On day 0 the antigen was injected i.v. intouninfected controls and into each of the infectedgroups. Four days later, i.e., days 28, 18, and 8postinfection, all the mice were killed, and theirspleens were assayed for PFC employing SRBCor SRBC coated with the respective antigen asdescribed above. The experimental data arerecorded in Table 1. In accordance with previ-ous findings (5, 27), the PFC responses againstSRBC were unaffected during the first week ofinfection but were reduced by 92 to 96% inanimals examined 18 and 28 days after virusinfection. With regard to the thymus-independ-ent antigens, experiments were performed induplicates. It may be noted that relatively fewPFC appeared in all mice sensitized with S IIIor PVP as compared with those that could beachieved with SRBC or LPS. To evaluate thedata, the Wilcoxon rank test was used to testthe PFC counts of infected groups versus thecorresponding control groups. The 0.05 signifi-cance level was reached in the case of twogroups sensitized with thymus-independent an-

tigens: one group showed a threefold increaseand the other showed a twofold reduction ascompared with the control groups (Table 1). Forthe evaluation of the results, however, therelatively large number of groups (24) should

TABLE 1. PFC responses against SRBC, LPS, SIII, and PVP in the course of acute LCM virus infectiona

PFC/spleen (x 10-2) on various days p.i. and 4 days postpriming2Antigen Expt

No virus Day 8 p.i. Day 18 p.i. Day 28 p.i.

SRBC 1 582 582 26c 48c(320-832) (352-832) (6-59) (15-115)

LPS 1 271 418 596 405(93-476) (219-652) (118-1601) (253-620)

LPS 2 677 829 611 620(428-936) (260-1235) (238-1005) (281-901)

S III 1 48 30 69 40(20-92) (14-62) (49-96) (25-52)

S III 2 30 31 29 90d(7-55) (16-52) (10-82) (52-178)

PVP 1 33 34 23 33(18-47) (17-50) (16-31) (10-60)

PVP 2 68 62 29 36d(29-93) (46-87) (15-38) (25-66)

a LCM virus (103 LD56) was given i.p.; 2 x 108 SRBC, 50 ,g of LPS, 0.5 ug of S III, or 0.5 ug of PVP wasgiven i.v.'Means and ranges of groups (each comprising five mice). p.i., Postinfection.('Significance at the 0.01 level relative to uninfected controls.d Significance at the 0.05 level relative to uninfected controls.

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also be considered. Taking this into account, itseems justified to conclude that the responsive-ness against the thymus-independent antigenswas either unaffected by the LCM virus infec-tion or, at most, very slightly influenced as

compared with the pronounced suppression ofthe anti-SRBC response.

To complete the report on the describedexperiments, it should be added that the devel-opment ofLCM infection after virus inoculationwas checked by demonstrating the appearance

of specific complement-fixing antibodiesagainst LCM virus antigen.

Cytotoxic responses of LCM virus-infectedmice against tumor allografts. In the follow-ing experiments the T cell-dependent cytotoxicresponse against allogeneic cell transplants wasexamined in LCM virus-infected mice. Groupsof six C,H mice were inoculated i.p. with 103LD,0 of virus 10 and 0 days before sensitization.On day 0 three uninfected C.H mice andone-half of the infected mice received 5 x 106DBA/2 mastocytoma cells by the i.p. route. Tendays later, that is days 20 and 10 postinfection,the mice were killed together with three normalC,H control mice, and the pooled spleen cellsuspensions from the six different groups were

tested for in vitro cytotoxicity against DBA/2target cells as described above. The data fromthe experiment, performed in duplicate, are

recorded in Table 2. It can be seen that theresults obtained with nonsensitized mice,whether infected or not, were almost identical.LCM virus which might contaminate the spleencell suspensions prepared from infected miceseemed, therefore, not to affect significantly thechromium-51 release from the target cells. It isapparent from the table that the cytoxic re-

sponses achieved in allografted infected micewere close to normal when tested on day 10postinfection but were severely suppressedwhen tested on day 20 postinfection.

It has been shown previously (5, 27) that theLCM virus-induced suppression of the anti-SRBC response is transient and that gradualrestoration takes place 1 to 2 months afterinfection. To further investigate the time course

of suppression of the cytotoxic responsiveness,groups of three C3H mice were infected withLCM virus at various intervals (Table 3). All theanimals were injected i.p. with 5 x 10' DBA/2mastocytoma cells on the same day, and 10 daysafter this allografting the mice were killed, andtheir spleen cells were assayed for cytotoxicityas mentioned above. From Table 3 it is evidentthat a decreased response could be observed as

early as day 15 postinfection, and that gradualrestoration occurred about 1 month after infec-tion.

TABLE 2. Cytotoxicity of spleen cells from LCMvirus-infected CsH mice against DBA/2 mastocytoma

cells

5'Cr release with spleen cellsobtained on days 10 and 20 p.i.'

Expt AllograftingrNo virus Day Day

10 p.i. 20 p.i.

1 None -4 - 1 85 x 106 DBA/2 107 94 21

cells i.p.2 None 6 6 6

5 x 10' DBA/2 75 79 11cells i.p. 76 _79_ 11

a Allografting of C,H mice was performed 10 daysbefore measurement of cytotoxic cells.

Expressed as (mean counts per minute of fiveparallel cultures minus mean counts per minutesreleased spontaneously) x 10- 2. Spleen cell pools wereobtained from groups of three mice. p.i., Postinfec-tion.

TABLE 3. Cytotoxic responses of LCM virus-infectedmice at various intervals after virus inoculation

"Cr release with spleen cells obtained on variousdays postinfection and day 10 postallografting'

No Day Day Day Day Day Day Dayvirus 10 15 20 24 30 38 49

72 67 0 5 33 33 39 67

a Expressed as (mean counts per minute of fiveparallel cultures minus mean counts per minutereleased spontaneously) x 10-2. Spleen cell pools wereobtained from groups of three mice. DBA/2 mas-tocytoma cells (5 x 106) were given i.p. (DBA/2mastocytoma cells were also used as target cells).

Lymphoid cell transfer experiments. In aprevious report (5) it was suggested that theLCM virus-induced immunosuppression mightbe accounted for by a simple numeric deficiencyof immunocompetent cells. However, in thelight of recent reports on Moloney virus-in-fected mice (14, 22), it seems to be urgent alsoto consider suppressor cell activity as a possi-ble explanation of the immune defects associ-ated with LCM virus infection. In an attempt todemonstrate suppressor cells in infected immu-nosuppressed mice, the spleen cells from suchmice were examined in the following transplan-tation assay. Pooled spleen cell suspensionswere prepared from 5 to 6 normal mice, andfrom similar numbers of mice inoculated with103 LD,0 of virus 14 days previously. Thencell preparations were made which containedthe following numbers of cells per 0.5 ml: (i)50 x 106 normal cells, (ii) 50 x 106 cellsfrom infected mice, and (iii) a mixture contain-ing 50 x 106 of each of these cells. These celldoses were injected i.v. into groups of 5 to 6

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recipient mice which had previously beentreated with 800 R of X rays. After transplanta-tion the recipients received 2 x 108 SRBC i.p.,and 6 days later their spleens were assayed forPFC. From the data recorded in Table 4 itappears that the donor cells from the infectedmice gave rise to significantly decreased re-sponses and moreover that the PFV numbersachieved with mixtures of infected and normalcells were close to the sum of PFC produced bythe separate cell fractions. The results, there-fore, made it unlikely that suppressor cellscould play any significant role for the sup-pressed adoptive responses seen in recipients ofcells from infected donors. The findings seemedto exclude the possibility that virus contamina-tion of the transferred cell suspension mighthave interfered with the results obtained. Bloodvirus titrations performed just before sacrificeshowed that the recipients of cells from infecteddonors might carry small amounts of virus,ranging from no trace to 102 LD50 per 0.03 ml,whether they received such cells alone or inmixtures with normal cells.The findings presented in this paper have

strongly suggested the T lymphocyte populationto be the primary target of the LCM virus-induced immunosuppression. This assumptionwas further tested in the final experiments.From donor groups comprised of eight to ninenormal mice and five mice injected with virus14 days previously, cell preparations were pro-duced which contained the following numbers

TABLE 4. Interaction between LCM virus-suppressedspleen cells and normal spleen cells in the anti-SRBCresponse after transfer to 800 R-irradiated recipients

PFC per recipient spleenCell transplantsa (x 10- 2)b p''

Expt 1 Expt 2

Normal spleen cells 289 208(50 x 106) (148-444) (105-290)

Suppressed spleen 62 41cells (50 x 106) (33-103) (2-83) <0.01

Normal spleen cells 337 287(50 x 106) + sup- (184-516) (235-375) > 0.05pressed spleencells (50 x 106)

a Spleen cell pools were obtained from groups of fiveto six normal mice and five to six mice inoculated i.p.with 103 LD50 of virus 14 days previously.

b After irradiation and i.v. transplantation therecipients received 2 x 108 SRBC i.p.; PFC werescored 6 days later and are given as means and rangesof groups of five to six recipients.

c Wilcoxon rank test versus recipients of 50 x 106normal spleen cells (each experiment tested sepa-rately).

of cells per inoculation dose: (i) 50 x 106 normalthymocytes, (ii) 50 x 106 spleen cells from in-fected donors, (iii) a mixture of 50 x 106 of eachof these cells, and (iv) 50 x 106 normal spleencells. The cell preparations were injected intogroups of five lethally irradiated mice, whichwere then sensitized with SRBC and tested6 days later for PFC as described above (Ta-ble 5). The numbers of PFC found in the re-cipients of thymus cells were minimal andwithin the background level. However, whenthymus cells were added to the inferior spleencell populations prepared from infected mice,they caused a significant restoration of theresponsiveness against SRBC. The PFCnumbers obtained with such mixtures wereclose to those produced by the normal spleencells. It is apparent that these results areconsistent with the assumption that the restric-tion of the immune capacity is caused by a Tcell deficiency, whereas they are inconsistentwith the possibility that the B cells should bethe limiting component.

DISCUSSIONFrom previous papers (5, 27) on the LCM

virus it is apparent that this virus can causepronounced suppression of the thymus-depend-ent humoral immune response against SRBCand human serum albumin. The inhibition isexpressed during the second week of the acuteinfection and persists for about 2 months. TheLPS, S III, and PVP, which were employed in

TABLE 5. Interaction between normal thymocytesand LCM virus-suppressed spleen cells in the

anti-SRBC response after transfer to 800 R-irradiatedrecipients

PFC/recipient spleen

Cell transplantsa (x 10-2)bExpt 1 Expt 2

Normal thymocytes 0.31 0.18(50 x 106) (0.05-0.65) (0.05-0.45) <0.01

Suppressed spleen cells 54 56(50 x 106) (42-68) (29-90) <0.02

Normal thymocytes 128 133(50 x 106) + sup- (68-220) (66-168)pressed spleen cells(50 x 106)

Normal spleen cells 178 166(50 x 10) (72-304) (54-384) >0.05

a Thymus and spleen cell pools were obtained from groupsof eight to nine normal mice and five mice inoculated i.p. with103 LDso of virus 14 days previously.MAfter irradiation and i.v. transplantation the recipients

received 2 x 106 SRBC i.p.; PFC were scored 6 days later andare given as means and ranges of groups of five recipients.

( Wilcoxon rank test versus recipients of 50 x 106 normalthymocytes plus 50 x 106 suppressed spleen cells (eachexperiment tested separately).

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the present work, have all been demonstrated tobe strictly thymus-independent antigens (1, 17,30). Surprisingly, it was found that when theseantigens were administered to mice at variousstages of the LCM virus infection they gave riseto humoral immune responses which were simi-lar to those found in normal mice. On the otherhand, the allograft responses of the infectedmice revealed a significant impairment whichwas temporally correlated to the hyporespon-siveness against the thymus-dependent anti-gens. The allograft responses, as tested by the invitro cytotoxicity technique employed, has beenclearly defined as a function mediated by Tcells without requirement for B cells or macro-phages (7, 13). The present findings, therefore,make it justified *to assume that the centralevent in the LCM virus-induced immunosup-pression is a disorder affecting the T lympho-cytes. The significant restoration in the celltransfer experiments of the immunological ca-pacity of spleen cells from infected animals bythe addition of normal thymus cells is consist-ent with this assumption.A number of other viruses are also known to

depress thymus-dependent immune functionsin animals (10, 32; for further references seebelow) and in humans (21, 23, 32). In mostcases, however, the available information isincomplete, and evaluations and generaliza-tions as regards the immunodepressive mech-anisms are, therefore, difficult to carry out.The immunosuppression induced by murine

oncorna viruses has been studied most exten-sively. The humoral immune responsiveness ofradiation leukemia virus-infected mice was re-cently investigated by Peled and Haran-Ghera(35). Their findings show convincing similarityto the present observations with the LCM viruswith regard to clearcut restriction of the viralimmunosuppression to thymus-dependent im-mune functions. Also, Friend leukemia and theMoloney sarcoma viruses are known to causedistinct depression of a variety of in vitrocorrelates of T cell immunity (14, 22, 29) butaffect only to a relatively limited degree re-sponses against bacterial somatic antigens (9,16). In the papers by Gorczynski (14) andKirchner et al. (22) on the Moloney virus it wasstrongly suggested that the immunosuppressiveactivity of this virus was mediated by a popula-tion of suppressor cells, probably belonging tothe monocyte-macrophage series. The conceptof suppressor cells (12, 41), however, seems notto afford any ready explanation of the presentfindings with the LCM virus. The cell transferdata recorded in Tables 4 and 5 clearly demon-strate that the immunosuppressed spleen cell

population from infected mice would not confersuppression to normal spleen or thymus cells.These experiments also argue against mech-anisms involving the elaboration of humoralimmunosuppressive factors, which have beensuggested to play a role in the antigenic compe-tition phenomenon (40).Very recently it has been claimed that T cells

may carry receptors for certain viruses, e.g.,measles, influenza, parainfluenza, and cyto-megalovirus (45; A. M. Denman, personal com-munication; H. F. McFarland, manuscript inpreparation). In relation to the present observa-tions, it may seem attractive to propose such Tcell receptors for the LCM virus as affording anexplanation of a selective destruction of the Tlymphocytes. However, the small frequency ofinfected lymphocytes as estimated in immuno-fluorescent studies (less than 5% [25]), thenoncytopathic nature of the LCM virus, and thedemonstration of almost intact immune func-tions in the heavily infected persistent LCMvirus carriers (33) make it rather unlikely thatthe LCM virus should act by any direct exten-sive damage to the T lymphocytes.

Certain parallel observations make it inter-esting to compare the LCM virus with the lacticdehydrogenase (LDH) virus, which is known todepress cell-mediated immunity (18). Whileboth these viruses may cause persistent infec-tions in mice, it is notable that their im-munosuppressive effects are largely confined tothe acute stage of infection. Shortly after virusinoculation they cause a dramatic decrease inthymocytes (5, 15, 37) and also reduce thenumber of lymphocytes in the peripheral thy-mus-dependent regions (15, 37). The studies onthe LDH virus indicate that this cellulardepression is mediated by the early productionof some intermediate soluble factor affectingthe T cell population, and that this factor is notadrenal cortical steroid (37). In the LCM virusinfection there is also some evidence for theexistence of an early intermediate depressivefactor. This evidence has come from the studyof hemopoietic precursor cells, which are se-verely suppressed in the early stage of the LCMvirus infection (4, 5). It was suggested that thefactor might have an analogus effect on lymph-oid precursor cells, causing inhibition of theirproliferation and differentiation and leading toa numeric deficiency of mature immunocompe-tent cells. In the light of the presenct findings,this hypothesis might be extended by postulat-ing a selective interference with the T lympho-cyte population. Such selectivity might be ex-plained by a preferential effect of the inhibitingfactor on T cell precursors, or perhaps more

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likely, by assuming a higher cellular turnoverrate w-thin the T cell population than withinthe B cells. The turnover rate of T and B cells isas yet not very well defined. Attempts toestimate the life-span of unprimed B cells havegiven conflicting results (39), and informationabout the rate of conversion of stem cells to Bcells is poor (31). As to the lymphoid cellswithin the thymus, their rapid turnover rate iswell known. With regard to the peripheral Tcells, it has become clear that different sub-populations exist, and that cooperative interac-tion between these is involved in immune reac-

tions (11, 38). The recent convincing demon-stration of peripheral T cell subsets endowedwith a short life-span (28, 34) may, therefore,afford support to the foregoing discussed hy-pothesis.

In connection with the present demonstrationof the T lymphocytes as the target of the LCMvirus-induced immunosuppression, it should beemphasized that there is strong evidence that Tcells play the central role in the recovery fromLCM virus infections, and also that they are

indispensable in the production of antibodiesagainst the virus (8, 26, 42; M. Volkert, K.Bro-J0rgensen, 0. Marker, B. Rubin, and L.Trier, manuscript in preparation). It may,therefore, be suggested that the suppressiveeffect described could be a significant event inthe induction of tolerant and persistent infec-tions with this virus. Moreover, we have re-

cently obtained strong evidence that the effectmay also account for a distinct transient im-pairment of the tumor resistance in acutelyLCM virus-infected mice (F. Guittler, K. Bro-J0rgensen, and P. N. J0rgensen, manuscript inpreparation).

ACKNOWLEDGMENTS

We wish to express our gratitude to B. Hoff-Clausen and K.Florentz for skillful technical assistance.

This work was supported by grants 511-571, 512-817,512-1184, and 512-1635 from Statens LaegevidenskabeligeForskningsrad, Denmark.

LITERATURE CITED1. Anderson, B., and H. Blomgren. 1971. Evidence for

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