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Measles virus-induced suppression of lymphocyte proliferation
CELLULARIMMUNOLOGY
116,367-381(1988)
Measles Virus-Induced Suppression of Lymphocyte Proliferation’ MARY SAN~I-IEZ-LANIER, PETER GUEIUN, LEROY C. MCLAREN, AND ARTHUR D. BAHBHHR& Deportments ofMicrobiology and Medicine, University of New Mexico, School ofMedicine, Albuquerque, New Mexico 87131 Received January 7.1988; acceptedJune IO, 1988 The mechanism by which measles virus in&& immunosuppression was investigated using an in vitro system employing phytobemagglutinin (PHA)-induced human peripheral mononuclear cell (PBMC) proliferation. At a muhipheity of i&&on of 1.0 or greater measles virus significantly inhibited (45%) the proliferation of PBMC. This inhibition was not due to an alteration in the k&ties of proliferation. PHA-stimulated PBMC were then infected with measles virus for 72 hr and kdiated (3200 rad) to prevent further proliferation. These infected, irradiated PBMC when added to fresh autologousPBMCeaused signiftennt inhibition oflymphoproliferation over a wide range of infeetedzfresh cell ratios (maximum inhibition seen at a 1:1 ratio, 85% inhibition). Vii recovered from the irradiated, infected e&s was lOO-fold lower than the virus titer needed to cause inhibition by direct addition of me&es virus. However, antibody to measles virus reversed the inhibition. Virus-tree supematant fluids from the infected kmdkted cells caused immunosuppression of the PHA maponse. This immunosuppressive material indueed by the measles virus was maximally produced after 72 hr and did not appear to require viral mph&ion. This factor was not pros&$&in E or interferon-a or -7. The production of such suppressive factors during viral infection may explain some of the profound immunosuppression seen in situations in which little or no infectious virus can be deteeted. Q 1988 AC&I& Pres$ Inc.
INTRODWTION
The ability of viruses to suppress immune function has attracted a great deal of interest, partially resulting from the discovery that the profound immunosuppression observed in acquired immune deficiency syndrome (AIDS) is due to a virus. The immunodeficiency associated with viral infections was first noted in 1908 when von Pirquet observed a temporary depression in the tubercuhn skin test response in individuals with measks ( I). His observation that measlescan suppressimmune function has since been confirmed and extended; however, in spite of years of research the mechanism of measles-induced immunosuppression remains poorly understood (2’ Supported by NIH CA24873 and RR08 139 and a p&oeWml fellowship (M.S.-L.) from the American Society for Microbiology. ’ To whom reprint requests should be addressed.
367 OOW8749/88 $3.00 Ckqy+tQ 1988byAcaddcRer,Inc. .4Urightsofreph&minanyfonmrrsemd.
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6). Additionally, the relevance of the immunosuppression to the diseaseprocess and outcome is not well understood. During in vivo infection, measles virus can replicate in lymphoid tissue and viral antigen is expressed on the surface of infected lymphocytes (both T and B cells) after mitogen stimulation in vitro (7, 8). A measles infection can result in a reduction in delayed hypersensitivity reactions as well as alter intracellular immunoglobulin levels and lymphokine production (9, 10). In vitro, monocytes, T cells (both helper and suppressor cells), and B cells can all be infected with the virus ( 11, 12). Such in vitro infections have resulted in inhibition of lymphocyte proliferation, lymphokine production, antibody production, and natural killer cell activity, as well as other lymphoid cell functions (12-l 7). This virus-induced alteration of immune function is thought to play a role in the pathogenesis of postmeasles morbidity as well as to alter specific immune responses such that virus dissemination and/or persistence is enhanced ( 16). Measles virus-induced inhibition of lymphocyte proliferation, an in vitro correlate of the cell-mediated immune response, was chosen as the model in the present study for examining viral modulation of immune function. This in vitro model is simple and the ability of the measles virus to suppress PHA-induced lymphocyte proliferation is well established (17, 18). Using this system we have shown that measlesvirusinfected peripheral blood mononuclear cells (PBMC) when added to fresh autologous PBMC inhibit lymphoproliferation. The virus recovered from these irradiated, infected cells could not account for the inhibition seen since the virus titer from the infected cells was 1OO-foldlower than the virus titer required to cause similar suppression. The virus-infected cells, however, did secretea immunosuppressive factor which did not appear to require cell proliferation and was not prostaglandin or interferona or -7. METHODS AND MATERIALS Virus. Measles virus of the Edmonston strain was grown in Vero cell monolayers which were maintained on Eagle’s minimal essential medium (MEM) supplemented with 10%fetal bovine serum (PBS). Suspensions of virus were harvested at maximum cytopathic effect by one cycle of freeze-thaw, followed by sonication of the suspension. This material was then centrifuged for 10 min at 1600 rpm to remove large cellular debris and then ultracentrifuged at 65,000g for 1 hr. The resulting viral pellet was resuspended in l/ 10 of the original volume in MEM with 10% PBS and frozen at -70°C in 10% dimethyl sulfoxide. Uninfected Vero cell monolayers were treated identically as controls for the measles virus preparations. Both the Vero control and the measlesvirus suspensions were mycoplasma free. Viral plaque assay. Plaque titrations were carried out on Vero cell monolayers by an adaptation of the method of Rapp (19). After absorption of the virus to the cells the monolayers were overlayed with 5 ml of 0.4% agarose in MEM containing 2% PBS. After a 5&y incubation monolayers were fixed with 10% formalin and stained with crystal violet. The titer of virus was expressedasplaque-forming units per milliliter (PFU/ml). Interferon assays. The assayfor interferon (IPN) was performed by the cytopathiceffect reduction assay(20). Serial dilutions of the samples to be tested were made in
MEASLES VIRUS IMMUNOSUPPRESSION
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96-well microtiter plates. In parallel, dilutions of an IFN standard were also made. These were added to WISH cell monolayers and after 24 hr the monolayers were challenged with vesicular stomatitis virus (VSV). The monolayers were then stained as described and the plates were read photometrically using a vertical light path automated spectrophotometer (Dynatech laboratories, VA). Prostuglundin assay. Prostaglandin E (PGE) was measured using the PGE kits from Seragen(Seragen Inc., MA) and by the methods of Rigler et al. (2 1). Preparation of peripheral blood mononuclear cell suspensions. Peripheral venous blood was drawn from normal individuals in preservative-free heparin. PBMC were separated by centrifugation on Ficoll-Hypaque gradients and washed three times with phosphate-buffered saline (PBS). Viability by trypan blue exclusion was always greater than 98%. Peripheral blood lymphocyte subsets. To obtain lymphocyte suspensions depleted of glass-adherent cells, the PBMC were passedthrough a glasswool column (22). The number of monocytes remaining in the preparation was determined using a benzidine dihydrochloride stain (23). T cells and non-T cells were separated by E-rosette formation with S-(2-amino ethyl) isothiouronium bromide (AET)-treated sheep erythrocytes as previously described (24). When rerosetted, the T-cell population always contained fewer than 1% contaminating cells. The non-T-cell population contained fewer than 5% contaminating E-rosette-forming cells. Supernutuntfluid production. PBMC, 0.8 X 106/ml, were incubated for 24 hr in 2.0 &ml phytohemagglutinin (PHA). For some experiments the PHA was omitted. The supernatant fluid (SN) was then discarded and the cells were infected with measlesvirus or mock infected. After a 72-hr incubation, the cells were pelleted by centrifugation and the SN was collected. The SN was ultracentrifuged at 152,OOOgfor 2 hr and assayed for infectivity. SN always contained less than 10 PFU/ml of measlesvirus. Infected cell cultures. The infected cell pellets from which the SN fluids were collected were then resuspended in RPM1 1640 and the PBMC were irradiated with 3300 rad using a 13’Cssource (Isomedix, Inc., NJ). This dosagerendered the PBMC incapable of significant thymidine uptake while retaining cell viability. After irradiation the PBMC were resuspended in fresh medium. Lymphocyte proliferation assays. All PBMC were cultured in RPM1 1640 medium supplemented with 10% FBS and penicillin and streptomycin. An optimal amount of PHA (2.0 pg/ml) was added. PBMC (1 X 105)were added to each well of a 96-well microtiter plate. Depending on the experiment, dilutions of either the viral suspension (or uninfected control) or the supematant fluids or the infected, irradiated cells were added. The total volume of each well was brought to 250 ~1. Plates were incubated for 66 hr, pulsed with 2.0 X IO-’ Ci of [3H]thymidine, and harvested 12-18 hr later on glasswool filters on a Skatron cell harvester (Skatron, Inc., VA). RESULTS The efict of measles virus on PHA-stimulated lymphocytes. Initially, experiments were carried out to establish the parameters by which measles virus inhibited PHAstimulated proliferation of lymphocytes. Figure 1 shows the concentration of measles virus necessaryto inhibit lymphoproliferation. At a multiplicity of infection (moi) of
370
SANCHEZ-LANIER ET AL. 70
F @
50
f-
40
“0 F
30
g
20
P g
60
10
I I
c 0.001
+ 0.01
MULTIPLICITY
I
I
0.1
1.0
I 10
OF INFECTION (MOI)
FIG. 1.Measlesvirus-inducedinhibition of lymphoproliferation.Measlesvirus, at increasingmultiplicitiesof infection, wasaddedto PBMC which werestimulatedwith PHA (2.0&nl). The percentageinhibition wasdeterminedby comparingthe meancpm of virus-treatedculturesto the meancpm of culturesto which correspondingdosesof control tluid wereadded.Eachpoint representsthe meanpercentageinhibition f SD for four experiments.Control uninfectedfluids did not causeinhibition.
1.Oor greater measlesvirus significantly (P < 0.000 1 by the Student &test) inhibited the proliferation of PBMC. Below an moi of 0.1 the inhibition became negligible. PBMC treated with Vero control fluids, that is, mock infected, did not causesignificant inhibition (data not shown). When suboptimal (0.5 &ml) concentrations of PHA were used to stimulate the PBMC, the percentageinhibition causedby measlesvirus was either higher than the inhibition obtained with optimal doses of PHA or it remained the same (data not shown). The inhibition of lymphoproliferation by measlesvirus did not merely alter the kinetics af the lymphocyte responseto PHA (Fig. 2). The PBMC were stimulated with PHA and infected with measlesvirus at an moi of 1.0, or mock infected with the Vero control, Cultures were then pulsed and harvestedevery 24 hr for the life of the culture. At a harvest time of 24 hr postinfection, the proliferation of the PBMC was too low to be measuredby the uptake of [3H~ymidine. By 48 hr postinfection, there was significant uptake of the [3HJthymidine and someinhibition of the proliferation of the infected PBMC. At 72,96, and 120hr postinfection there was significant (P < 0.001 by the Student t test) inhibition by measlesvirus of lymphoproliferation. Despite the inhibition observed,cell viability was always greater than 85% and in most caseswasgreaterthan 95%. The e$ct of infected cells on PHA-stimulated lymphocytes. PHA-stimulated lymphocyteswere infected with measlesvirus or mock infected for 72 hr. The cells were then irradiated with 3200 rad which wasshown to prevent further proliferatian while retaining cell viability and added to fresh PHA-stimulated autologousPBMC at varying ratios of infected, irradiated cells to fresh cells The resulting proliferation of the freshPBMC is shown in Fig. 3. Inhibition causedby the measlesvirus-infected,irradiated lymphocytes of the proliferative responseof the fresh PBMC was highly significant (P K O.,OOl)at ratios ranging from 1:1 to 1:128 infected PBMC to fresh PBMC. The greatestamount of inhibition was seenwhen there was equal numbers of fresh lymphocytes ( 1 X 105)and irradiated lymphocytes ( 1 X 10s)in eachwell ( f : 1). How-
MEASLES VIRUS IMMUNOSUPPRESSION
16 TIME OF CULTURE
371
120
HARVEST
(hours after infection)
FIG. 2. The kinetics of measles-induced inhibition of lymphoproliferation. Peripheral blood lymphocytes were stimulated with PHA (2.0 &ml) infected with measles virus and pulsed and harvested over a 5-day period. The data are expressedas mean cpm + SD for triplicate cultures from one donor and are representative of three experiments. There was no signi$cant difference in cell viability between control and infected cultures at any time.
64
66
46
16.
6-
FIG. 3. The inhibitory effect of irradiated, measles virus-infected peripheral blood lymphocytes on the response of fresh autologous PBMC to PHA. Lymphocytes which were either mock infected or infected with measles virus and then irradiated with a dose that prevented further proliferation were added in varying numbers to fresh autologous PBMC. The proliferative response of the fresh PBMC to PHA was measured by uptake of [3H]thymidme. Results are expressedas mean f SD for five experiments. (0) Mockinfected PBMCResh PBMC. (0) Measles virus-infected PBMCResh PBMC.
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WV moi=o.o1
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12345071 Donor moi.o.01
Mv moi.l.0
FIG. 4. The inhibitory effects of measles virus compared to the inhibitory effects of irradiated, infected PBMC on lymphoproliferation. Measles virus at an moi of either 0.01 or 1.Owas added directly to lymphocytes which had been stimulated with PHA. Irradiated, infected lymphocytes were assayed for infectious measlesvirus by plaque assayand added to fresh autologous PBMC at a concentration that established an moi of 0.01. For each donor reported cultures were done in triplicate and the variance was less than 10%. The two bars representing measlesvirus added directly are the four experiments as reported in Fig. 1.
ever, significant inhibition was still observed with some donors when there was only one irradiated, infected lymphocyte for every 512 fresh autologous lymphocytes. Lymphocytes which had been mock infected and irradiated did not affect the proliferation of the fresh autologous PHA-stimulated lymphocytes. The viability of the irradiated cells was greater than 92% for the life of the culture so the inhibition was not due to cell death. The PBMC which had been irradiated were all plaque assayedfor the presence of infectious measlesvirus. The cells were assayedby directly absorbing them to the cell monolayer for 1.5 hr or by sonicating them first for cellular disruption. No appreciable difference was found in the amount of infectious virus using the two different methods. Figure 4 compares the inhibition of PHA-induced proliferation caused by direct addition of measles virus to the inhibition caused by infected, irradiated lymphocytes. The infected, irradiated PBMC which had been assayedfor infectious virus were added in ratios ranging from 1:16 to 1:64, infected to fresh PBMC, such that measlesvirus was added at an moi of 0.0 1. Direct addition of measlesvirus at an moi of 0.0 1 caused substantially less inhibition. Measles virus added at an moi of 1.O caused comparable or slightly less inhibition. Therefore, inhibition caused by the measlesvirus-infected, irradiated PBMC was greater than that caused by direct addition of measles virus and at an moi which was lOO-fold lower than that needed to cause inhibition by direct addition of measlesvirus. Measles virus is known to undergo substantial replication only in PBMC which have been stimulated with PHA. Supematant fluids collected from PBMC which had been infected with measles virus in the absence of PHA contained fewer than 10 PFU/ml. However, if these same PBMC were then stimulated with PHA, even 96 hr postinfection, substantial amounts of virus could be obtained in the SN. Thus it was necessaryto determine whether or not the lymphocytes needed to be stimulated with PHA prior to infection with measlesvirus in order to causeinhibition of lymphoproliferation. Figure 5 shows the results of an experiment where the PBMC were either
MEASLES VIRUS IMMUNOSUPPRESSION
Control
1:4
cl
UNSTWJATED INFECTED IRRMIAITD PBL
o
WA
S”YULA,EC
1:8
1:16
373
INFECTED
1:32
FIG. 5. A comparison of the effect of stimulated and unstimulated measles virus-infected, irradiated PBMC on lymphoproliferation. Lymphocytes were incubated in the presence or absence of PHA for 24 hr. They were then infected with measles virus. The ratios expressed on the abscissa are the ratios of measles-infected, irradiated PBMC to fresh PBMC.
stimulated with PHA or left unstimulated for 24 hr and then either infected with measles virus or mock infected. After 72 hr in the absence of PHA the PBMC were irradiated and added to autologous PBMC to determine whether substantial replication of measles virus in the PBMC prior to irradiation was necessaryto observe the inhibition. All infected PBMC regardless of whether they had been stimulated with PHA prior to infection were inhibitory, although the inhibition causedby the unstimulated infected PBMC was significantly less than that caused by stimulated infected cells. Therefore, it was not necessaryfor the virus to undergo multiple cycles of replication in the lymphocytes prior to the addition of fresh lymphocytes, for inhibition to be observed. In spite of the fact that substantially lessvirus could be cultured from measlesvirusinfected PBMC than the amount of virus needed to suppress lymphoproliferation it was still possible that the measlesvirus and not the cells was suppressing the lymphoproliferation. Therefore, antibody to measles virus was added to cultures of PHAstimulated lymphocytes and to virus-infected irradiated PBMC. Two sources of antimeaslesvirus antibody were used (Table 1). Autologous serum from measles-seropositive donors was shown to partially reverse the inhibition caused by the infected PBMC at the ratios shown in Table 1. However, by plaque titration the autologous serum did not totally neutralize the infectious virus. Higher titered serum from patients with subacute sclerosing panencephalitis (SSPE) was also used. (Serum was a gift from Larry E. Davis, Department of Neurology, University of New Mexico, School of Medicine.) Serum from several patients were used to avoid obtaining results which might be associated with a particular patient. A range of serum dilutions were used so that the concentration of serum varied from that dilution of serum which just neutralized infectious virus (usually a 150 dilution of serum) to an excess of neutralizing antibody. All cultures were checked by plaque titration to assure that all virus was neutralized. From Table 1 it can be seen that the SSPEserum completely reversed the inhibition caused by the irradiated, measles virus-infected cells. The
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TABLE 1 The Effect of Antibody to Measles Virus on the Inhibition of Lymphocyte Proliferation Caused by Measles Virus-Infected Lymphocytes % Inhibition Donor serum*
Infected:uninfected”
No serum
Autologous serum
SSPE
1:6 1:16 1:8 1:16 I:16 1:32
82 72 91 88 79 71
49 38 55 46 ND’ ND
6 18 10 16 1 5
1 2 3
a Measles virus-infected irradiated lymphocytes were added in the indicated ratios to fresh PHA-stimulated lymphocytes. * Sera from patients with subacute sclerosing panencephalitis were added so that all infectious measles virus was effectively neutralized. ’ Not done.
SSPEserum was then absorbedwith measlesvirus-infected Vero cellsand the serum added to cultures of infected irradiated PBMC and PHA-stimulated PBMC. The absorption of the SSPEserum was verified by the elimination of immunofluorescent staining of measles-infectedVero cells.The inhibitory effectsof the cellswere significantly although not completely restored (Table 2). As an additional control, autologous and SSPEserawere added to cultures of PHA-stimulated PBMC to which measlesvirus (moi = 1, 5) was added directly. The SSPEserum completely neutralized the infectious virus and reversedthe inhibitory effectsof the virus. The autologous serum did not completely neutralize the virus and only partially reversedthe inhibitory effectsof the virus (data not shown). TABLE 2 The Effect of Absorbing Out Antibody to Measles Virus from SSPE Serum on the Inhibition of Lymphocyte Proliferation Caused by Measles Virus-Infected Lymphocytes 90Inhibition
Infecteduninfected” 1:4 1:s 1:16
No serum
SSPEserum *
Mock absorbed serumC
Absorbed serumd
91 87 83
6 6 7
8 16 12
42 43 43
0 Measles virus&fected irradiated lymphocytes were added at the indicated ratios to fresh PHA-stimulated lymphocytes. * Serum from patients with subacute sclerosing panencephalitis was added so that all infectious measles virus was effectively neutmlized. c SSPEserum was absorbed using mock-infected Vero celIs. d SSPEserum was absorbed using measlesvirus-infected Vero cells.
MEASLES VIRUS IMMUNOSUPPRESSION
i
375
1
TIME OF CCUECTION OF SUPERNATWTS hJn oner i7fectioil) FIG. 6. The inhibitory effectsof supematant fluids on PHA-induced proliferation of lymphocytes. Supernatant fluids were collected from cultures of lymphocytes at 24-hr intervals following infection with measlesvirus or mock infection. Data are expressed as mean cpm 2 SD for triplicate cultures.
The efict of supernatant fluids on lymphoproliferation. Another possible mechanism for the measlesvirus-induced immunosuppression is the production of suppressive soluble substancesby the measlesvirus-infected lymphocytes. SN were collected from measlesvirus-infected and mock-infected PBMC at 24&r intervals. After ultracentrifugation the SN were assayedfor infectivity and found to contain less than 10 PFU/ml. The virus-free SN were added to fresh PBMC stimulated with PHA at a dilution of I:5 or 1:10. The results of these experiments are shown in Fig. 6. Supematant fluids collected from PBMC immediately following infection with measlesvirus (0 hr) or 24 hr later did not significantly affect the response of PBMC to PHA. The SN collected at later times from measlesvirus-infected PBMC were inhibitory when compared with SN collected from mock-infected controls. Supematant fluids collected from PBMC which had been treated with Vero control material (mock infected) were not generally inhibitory to mitogenesis when compared to the untreated control (data not shown). For all subsequent experiments, SN were collected from measles virus-infected PBMC 72 hr after infecting the cells. The inhibitory potential of the SN collected at this time showed some donor variation. When optimal concentrations of PHA (2.0 &ml) were used, the mean percentage inhibition caused by the SN was 39%. In the cases of suboptimal concentrations of PHA (0.5 &ml), the mean percentage inhibitions was 60%. It was then of interest to determine whether infected PBMC must produce infectious virus in order to produce the suppressive SN. The PBMC were either stimulated with PHA for 24 hr or left unstimulated, then infected with measles virus or mock infected, and the SN were collected after 72 hr as usual. Table 3 compares the effect that SN collected from both stimulated and nonstimuIated PBMC had on lymphoproliferation. Both sets of SN were inhibitory; thus production-of infectious virus partic& was not a prerequisite to produce suppressive SN. Verification of viral infection of stimulated PBMC was made by detectionof virus by plaque assay.
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TABLE 3 The Effect of PHA Stimulation on the Production of Suppressive Supematant Fluids by Measles Virus-Infected Lymphocytes Unstimulated”
Donor 1 2
Vero control 83,726’ (12,876) 40,181 (795)
PHA stimulated*
virus
Vero
virus
infected
% Inhibition
control
infected
% Inhibition
48,373 (2,586) 28,568
42
79,463 (4,390) 38,799
39,019 (2,766) 25,954
51
(2,223)
(1,369)
(2,795)
29
33
’ PBMC were incubated with media for 24 hr prior to either mock infecting or infecting with measles virus. The SN were collected after 72 hr and added to fresh PBMC. b PBMC were stimulated with PHA for 24 hr prior to either mock infecting or infecting with measles virus. The SN were collected after 72 hr and added to fresh PBMC. c Data are expressed as mean cpm (SD) for triplicate cultures.
The role of lymphocytesubsetsin the production of the suppressiveSN. In order to determine which subsets of PBMC might be responsible for producing the suppressive soluble substance various cell subpopulations were assayed.First, the adherent cell population, consisting primarily of monocytes, was removed from the PBMC suspensions by passing the lymphocytes over glasswool columns. The resulting nonadherent cell population (~2% monocytes by histochemical criteria) was stimulated with PHA for 24 hr and then infected with measlesvirus or mock infected. After 72 hr the SN were collected and added to fresh, PHA-stimulated PBMC. Supematant fluids always contained fewer than 10 PFU/ml. There was no difference in the inhibition caused by SN from nonadherent lymphocytes when compared to the inhibition caused by SN from the unfractionated PBMC (data not shown). The lymphocytes were further fractionated into E-rosette-forming cells (T cells) and non-E-rosetteforming cells (non-T cells) by rosette sedimentation with (AET)-treated sheep erythrocytes. Both of these subpopulations of PBMC were stimulated and infected with measlesvirus and the SN collected after 72 hr. From the data presented in Table 4, it is apparent that the suppressive soluble substance can be obtained from both populations of cells and the inhibition was similar to that caused by SN collected from unfractionated, infected PBMC. Lack of a role for prostaglandins and interferon in the SN. Prostaglandins, which are known to suppresslymphoproliferation, might be contained in the SN and cause the suppression. To examine this, indomethacin (1 pg/ml), a prostaglandin synthetase inhibitor, was added to measles virus-infected and mock-infected PBMC and supematant fluids were collected after 72 hr of incubation. The SN which had been collected from PBMC treated with indomethacin were as inhibitory as SN collected when indomethacin was not present, suggestingthat prostaglandins were not responsible for the inhibition caused by the SN (data not shown). More direct evidence that prostaglandins were not involved in the suppression caused by the SN was found by measuring the amount of PGE in the SN. PBMC were stimulated with PHA and either infected with measlesvirus or mock infected. Supematant fluids were collected
377
MEASLES VIRUS IMMUNOSUPPRESSION TABLE 4 A Comparison of the Elfect of Supematam Fluids Collected from E-Rosette-Forming Cells and Non-E-Rosette-Forming Cells on Proliferating PBMC Supematant fluids” Donor
Virus-infected*
I
-
Unfractionated
+
114,703e (3,114) 69,155
-
97,512
+
70,435 (2,224) 81,110 (3,494) (47,325) (1,748)
(2,454) 2
(3,442) 3
-
+
E-RFC’
Non-E-RFCd
116,721 (5,918) 61,982 (326) 82,191 ( 1,204) 57,744
124,284 (3,918) 103,640 (5,222) ND/
78,150 (2,831) 49,888
58,707 ( 1,069) 83,818 ( 1,842) 47,73 1
(602)
(2,225)
(2,055)
’ Each of the three populations of lymphocytes was stimulated with PHA and either mock infected or infected with measlesvirus, and supematant fluids were collected 72 hr later. b PBMC were either mock infected (-) or infected with measlesvirus (+). c E-rosette-forming cells contained fewer than 1%contaminating cells when rerosetted. d Non-E-rosette-forming cells contained fewer than 5% contaminating E-rosette-forming cells. ’ Data are expressed as mean cpm (standard deviation) for triplicate cultures. ‘Experiment not done.
24, 48, and 72 hr postinfection and frozen at -70°C for the prostaglandin assay. Supematant fluids collected from measlesvirus-infected PBMC did not differ significantly from SN collected from the mock-infected lymphocytes in their prostaglandin content. Since viral-induced IFN is known to inhibit lymphoproliferation the amount of IFN contained in the SN collected from infected and mock-infected PBMC was directly measured by biological assay.The concentrations of IFN were donor variable, but in caseswhere the same donor was used several times, that donor was found to be consistent. Most SN from measles virus-infected PBMC ranged from 100 to 500 units/ml of IFN. Anti-EN antibody to either EN-(Y or IFN-r (Interferon Sciences, Inc., NJ) was added to the SN from both measles virus-infected and mock-infected PBMC. At the same time, the IPN antibody was added to known samples of IFN to serve as a control. Both the antibody-treated SN and the antibody-treated IFN-a control were added to PBMC which had been stimulated with PHA and the incorporation of [3H]thymidine was measured. The results of these experiments are shown in Tables 5 and 6. The anti-IFN-a! completely reversed the inhibitory effectsof the IFN(Yon the proliferating PBMC (Table 5). However, the anti-IFN-cl! had no effect on the SN collected from the measles virus-infected PBMC such that, with or without the IFN antibody, the SN was still inhibitory (Table 6). Similar results were obtained when antibody to IFN-7 was added to the SN (data not shown). Thus, antibodies to either IF%(u or IFN-y had no effect on the inhibitory effects of SN. The antibody-
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TABLE 5 The Effect of Interferon-a and Antibody to Interferon-a on Proliferating PBMC IFN--y + antibody to 1FN-y b IPNq (units) Donor
PHA”
500
100
15
1
2.0 0.5 2.0 0.5
31’ 63 30 71
28 57 30 67
18 48
2.0 0.5 2.0 0.5
35 72
35 62 27 37
20 44
2 3 4
ND ND
25 50
10 26
2000 units IPN--y +2OOO+NUAb
500 units 1FN-r + 1OOONUAb
0 0 0 0 NDd ND ND ND
0 0 0 0 ND ND ND ND
@Concentration PI-IA in &ml. ‘Antibody to IFN (neutralizing units/ml) completeIy neutralixed the active IPN-a as measured by plaque reduction assay. ’ Data are expressed as percentage inhibition of media controt d Not done.
TABLE 6 The Effect of Antibody to Interferon-a on Inhibitory Supernatant Fluids Antibody to IFN-o (neutralizing units/mI)“ Donor
PHAb
None
5ooo
2000
loo0
500
1
2.0 0.5 2.0 0.5 2.0 0.5 2.0 0.5 2.0 0.5 2.0 0.5
23” 50 29 54 40 42 20 83 38 64 37 54
NDd ND 25 50 ND ND ND ND ND ND ND ND
22 52 16 31 ND ND <5 69 ND ND ND ND
22 55 23 45 48 40 <5 73 43 65 37 63
18 57 ND ND ND ND <5 74 ND ND ND ND
2 3 4 5 6
a Ail concentrations of antibody to IPN completely neutral&d the active 1PN-u as measured by a plaque reduction assay. bConcentration PI-IA in &ml. ’ Data are expressed as percentage inhibition of media control. dNotdone.
MEASLES VIRUS IMMUNOSUPPRESSION
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containing SN were also assayedby biological assayfor IFN and there was no detectable IFN in the SN. Finally, inhibitory SN collected from PBMC which had been infected but not prestimulated with PHA contained little detectable IFN. These data would indicate that IFN is not the suppressive agent in the SN. DISCUSSION Even though numerous laboratories have investigated the mechanisms responsible for measles virus-induced suppression, it remains poorly understood. The suppression may be due to a direct effect of the virus on cell proliferation and to a viralinduced change in the function of the infected lymphocyte. This study was designed to investigate the latter possibility. First, it was shown that measlesvirus induced the release of a suppressive soluble factor. The suppression caused by this soluble factor was not due to measlesvirus, PGE, or IFN. Second, production of infectious virus was not required to produce the suppressive soluble factor. Third, measlesvirus-infected lymphocytes were able to suppress lymphocyte proliferation at a concentration of virus which was lOO-fold lower than the concentration of measles virus usually required to induce such suppression; yet this suppressive activity could be blocked by measlesvirus antibody. The literature contains conflicting reports regarding the role of a suppressive soluble factor in measlesvirus-induced immunosuppression. When such a factor has been found, its properties vary with the report. For example, measlesvirus-infected HeLa cells produced a soluble factor suppressive to DNA synthesis (25). The production of that particular soluble factor required infectious virus. Neighbour and Bloom found that SN from measles virus-infected lymphocytes were suppressive to concanavalin A responses.This suppressive activity was attributed to IFN (26). Salmi et al. found that SN collected from measlesvirus-infected lymphocytes were also suppressive (27). In this case,antibodies to IFN abolished the suppression only partially suggestingthat other factors contributed more to the suppression than IFN. Hirsch et al. found a suppressive soluble factor in 9 of 20 SN collected from PBMC of measles patients and cultured in the absence of any stimulant (28). This suppressive substance was not further characterized. In patients with acute measles, the [‘H]thymidine uptake of lymphocytes with either PHA or candida antigen was lower in media containing autologous serum than in media containing FBS (29). This soluble inhibitor also was not further characterized. Two previous reports were unable to find any suppressive activity in SN generated from measlesvirus-infected PBMC ( 17,30). One reason for the differences might be in the Edmonston strain of virus used. Volckaert-Vervliet et al. reported that different strains of measles virus, and even virus of the same strain but from different sources,induced widely variable amounts of IFN (3 1). It is conceivable that this strain variation could apply to other soluble substances in addition to IFN. While the above reports on the presence of a suppressive soluble factor were often conflicting, the present study brings up several interesting points. Similar to the studies of Hirsch et al. some variation in the ability of different donor lymphocytes to produce a suppressive soluble substance was observed. It is conceivable that such variation in the ability to produce a suppressive factor might be extrapolated to differences in patients who get complications of measles. The present report showed
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for the first time that measles virus contained within unstimulated PBMC could induce a suppressive soluble factor. Since only a limited number of lymphocytes in the body respond to a measlesinfection with proliferation, and the majority of lymphocytes in the circulation are in a resting stage, this is highly significant. An infected lymphocyte does not need to be proliferating in order for the virus to alter lymphocyte function. This also means that a small number of infected cells producing such suppressive factors can have an effect on other uninfected lymphocytes or even other types of cells. This provides another mechanism by which the virus could aid in its own dissemination and persistence as well as facilitate secondary infections. The present study showed that the amount of infectious virus recovered from sup pressive irradiated cells was significantly lower than the titer of virus required to induce suppression in fresh cells. In spite of this, the suppression still appeared to be due to measles virus since antibody to the virus reversed the suppression. There are several possible explanations. The tirst possibility is that some kind of temperaturesensitive or other type of mutant was developed in culture but not measured by the standard plaque assay.In this case,enough virus would be present to causethe inhibition but it simply was not measured by the assay.Second, the virus could also induce a suppressive soluble factor. The present study did show that such a mechanism is operative during measles infection although it is unclear if the production of such a factor can entirely explain the phenomenon addressed above. A third possibility is that infection of the lymphocytes by the virus altered the properties of the measles virus making it far more suppressive. Such a mechanism might explain why in the case of some viral infections the immunosuppressive effects are high while the amount of virus present is low. Lastly, infection of the fresh, PHA-stimulated lymphocytes may be more efficient when the virus is cell associated. This does not seem likely due to the long replication cycle of the virus, the relatively short term of the culture, and the low ratios of infected cells to uninfected cells. In addition, when measlesvirus is added to PBMC cultures at an moi of 10 so that all cells are infected, the percentage inhibition is never greater than 80% which is lower than the maximum inhibition obtained in this study. In summary, measlesvirus has been shown to inhibit lymphocyte proliferation by the induction of a suppressive soluble factor and by suppressive infected, irradiated cells. The production of such suppressive factors during viral infection may explain some of the profound immunosuppression seen in situations in which little or no infectious virus can be recovered. ACKNOWLEDGMENTS The authors thank Mary Padilla for her help in preparing the manuscript, Stella Falter and Anita Follins for technical assistance,and Michael McChesney, M.D., for his helpful suggestions.
REFERENCES 1. 2. 3. 4. 5. 6.
Von Pirquet, Dtsch. Med. Wochenschr.34,1297, 1908. Mellman, W. J., and Wetton, R., J. Lab. Clin. Med. 61,453, 1963. Hiklreth, E. A., Frederic, M. W., and Randall, P., Trans. Amer. C/in. Climatol. Assoc. 75,37, 1963. Brody, J. A., Overfield, T., and Hammes, L. M., N. Engl. J. Med. 271, 1294, 1964. Fireman, P., Friday, G., and Kumate, J., Pediatrics 43,264, 1969. Starr, S., and Berkovich, S., N. Engl. J. Med. 270,386, 1964.
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1. White, R. G., and Boyd, J. F., Clin. Exp. Immunol. 13,343, 1973.
8. Osunkoya, B. O., Adeleye, G. I., Adejumo, T. A., and Sahmonu, L. S., Arch. Ogesamte Virusforshung 44,323, 1974. 9. Coovadia, H. M., Parent, M. A., Loening, W. E. K., Wesley, A., Burgess, B., HaIlett, F., Brain, P., Grace, J., Naidoo, J., Smythe, P. M., and Vos, G. H., Amer. J. Clin. Nutr. 27,665, 1974. 10. Joffe, M. I., and Rabson, A. R., Clin. Immunol. Immunopathol. 20,2 15, 1981. 11. Joseph, B. S., Lampert, P. W., and Oldstone, M. B. A., J. Virol. 16, 1638. 12. Huddlestone, J. R., Lampert, P. R., and Oldstone, M. B. A., C/in. Zmmunol. Zmmunopathol. 15,502, 1980. 13. Casali, P., Rice, G. P. A., and Oldstone, M. B. A., J. Exp. Med. 159, 1322, 1984. 14. Pelton, B. K., Hylton, W., andDenman, A. M., Clin. Exp. Immunol. 47, 19, 1982. 15. McChesney, M. B., Fujinami, R. S., Lampert, P. W., and Oldstone, M. B. A., J. Exp. Med. 163, 1331, 1986. 16. Galama, J. M. D., Ubels-Postma, J., Vos, A., and Lucas, C. J., Cell. Zmmunol. 50,405, 1980. 17. Sullivan, J. L., Barry, D. W., Albrecht, P., and Lucas, S. J., J. Immunol. 114, 1458, 1975. 18. Lucas, C. J., Galama, J. M. D., and Ubels-Postma, J., Cell. Immunol. 32,70, 1977. 19. Rapp, T., J. Bacterial. 88, 1448, 1964. 20. Yeh, T.-J., McBride, P. T., Overall, J. C., Jr., and Green, J. A., J. Clin. Microbial. 16,4 13, 1982. 21. Rigler, G. L., Peake, G. T., and Ratnee, A., J. Endocrinol. 70,285, 1976. 22. Greaves, M., and Brown, G., J. Immunol. 112,420,1914. 23. Kaplow,L. S., Blood26,215, 1965. 24. Pellegrino, M. A., Ferrone, S., Dierich, M. P., and Reisfeld, R. A., Clin. Immunol. Immunopathol. 3, 324, 1975. 25. Minegawa, T., Nakaya, C., and IbA, H., J. Virol. 13, 118, 1974. 26. Neighbour, P. A.,and Bloom, B. R., Proc. Natl. Acad. Sci. USA 76,476, 1979. 27. Salmi, A., Vydelingum, S., Salonen, R., and Ilonen, J., Fed. Proc. 44,15 18,1987. 28. Hirsch, R. L., Griffin, D. E., Johnson, R. T., Cooper, S. J., DeSoriano, I. L., Roedenbeck, S., and Vaisberg, A., Clin. Immunol. Immunopathol. 31, 1, 1984. 29. Whittle, H. C., Dossetor, J., Oduloju, A., and Bryceson, A. D. M., J. Clin. Invest. 62,678, 1978. 30. Lucas, C. J., Ubels-Postma, J., Galama, J. M. D., and Rezee, A., Cell. Zmmunol. 37,448, 1978. 3 1. Volckaert-Vervliet, G., Heremans, H., de Ley, M., and Billiau, A., J. Gen. Virol. 41,459.
116,367-381(1988)
Measles Virus-Induced Suppression of Lymphocyte Proliferation’ MARY SAN~I-IEZ-LANIER, PETER GUEIUN, LEROY C. MCLAREN, AND ARTHUR D. BAHBHHR& Deportments ofMicrobiology and Medicine, University of New Mexico, School ofMedicine, Albuquerque, New Mexico 87131 Received January 7.1988; acceptedJune IO, 1988 The mechanism by which measles virus in&& immunosuppression was investigated using an in vitro system employing phytobemagglutinin (PHA)-induced human peripheral mononuclear cell (PBMC) proliferation. At a muhipheity of i&&on of 1.0 or greater measles virus significantly inhibited (45%) the proliferation of PBMC. This inhibition was not due to an alteration in the k&ties of proliferation. PHA-stimulated PBMC were then infected with measles virus for 72 hr and kdiated (3200 rad) to prevent further proliferation. These infected, irradiated PBMC when added to fresh autologousPBMCeaused signiftennt inhibition oflymphoproliferation over a wide range of infeetedzfresh cell ratios (maximum inhibition seen at a 1:1 ratio, 85% inhibition). Vii recovered from the irradiated, infected e&s was lOO-fold lower than the virus titer needed to cause inhibition by direct addition of me&es virus. However, antibody to measles virus reversed the inhibition. Virus-tree supematant fluids from the infected kmdkted cells caused immunosuppression of the PHA maponse. This immunosuppressive material indueed by the measles virus was maximally produced after 72 hr and did not appear to require viral mph&ion. This factor was not pros&$&in E or interferon-a or -7. The production of such suppressive factors during viral infection may explain some of the profound immunosuppression seen in situations in which little or no infectious virus can be deteeted. Q 1988 AC&I& Pres$ Inc.
INTRODWTION
The ability of viruses to suppress immune function has attracted a great deal of interest, partially resulting from the discovery that the profound immunosuppression observed in acquired immune deficiency syndrome (AIDS) is due to a virus. The immunodeficiency associated with viral infections was first noted in 1908 when von Pirquet observed a temporary depression in the tubercuhn skin test response in individuals with measks ( I). His observation that measlescan suppressimmune function has since been confirmed and extended; however, in spite of years of research the mechanism of measles-induced immunosuppression remains poorly understood (2’ Supported by NIH CA24873 and RR08 139 and a p&oeWml fellowship (M.S.-L.) from the American Society for Microbiology. ’ To whom reprint requests should be addressed.
367 OOW8749/88 $3.00 Ckqy+tQ 1988byAcaddcRer,Inc. .4Urightsofreph&minanyfonmrrsemd.
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6). Additionally, the relevance of the immunosuppression to the diseaseprocess and outcome is not well understood. During in vivo infection, measles virus can replicate in lymphoid tissue and viral antigen is expressed on the surface of infected lymphocytes (both T and B cells) after mitogen stimulation in vitro (7, 8). A measles infection can result in a reduction in delayed hypersensitivity reactions as well as alter intracellular immunoglobulin levels and lymphokine production (9, 10). In vitro, monocytes, T cells (both helper and suppressor cells), and B cells can all be infected with the virus ( 11, 12). Such in vitro infections have resulted in inhibition of lymphocyte proliferation, lymphokine production, antibody production, and natural killer cell activity, as well as other lymphoid cell functions (12-l 7). This virus-induced alteration of immune function is thought to play a role in the pathogenesis of postmeasles morbidity as well as to alter specific immune responses such that virus dissemination and/or persistence is enhanced ( 16). Measles virus-induced inhibition of lymphocyte proliferation, an in vitro correlate of the cell-mediated immune response, was chosen as the model in the present study for examining viral modulation of immune function. This in vitro model is simple and the ability of the measles virus to suppress PHA-induced lymphocyte proliferation is well established (17, 18). Using this system we have shown that measlesvirusinfected peripheral blood mononuclear cells (PBMC) when added to fresh autologous PBMC inhibit lymphoproliferation. The virus recovered from these irradiated, infected cells could not account for the inhibition seen since the virus titer from the infected cells was 1OO-foldlower than the virus titer required to cause similar suppression. The virus-infected cells, however, did secretea immunosuppressive factor which did not appear to require cell proliferation and was not prostaglandin or interferona or -7. METHODS AND MATERIALS Virus. Measles virus of the Edmonston strain was grown in Vero cell monolayers which were maintained on Eagle’s minimal essential medium (MEM) supplemented with 10%fetal bovine serum (PBS). Suspensions of virus were harvested at maximum cytopathic effect by one cycle of freeze-thaw, followed by sonication of the suspension. This material was then centrifuged for 10 min at 1600 rpm to remove large cellular debris and then ultracentrifuged at 65,000g for 1 hr. The resulting viral pellet was resuspended in l/ 10 of the original volume in MEM with 10% PBS and frozen at -70°C in 10% dimethyl sulfoxide. Uninfected Vero cell monolayers were treated identically as controls for the measles virus preparations. Both the Vero control and the measlesvirus suspensions were mycoplasma free. Viral plaque assay. Plaque titrations were carried out on Vero cell monolayers by an adaptation of the method of Rapp (19). After absorption of the virus to the cells the monolayers were overlayed with 5 ml of 0.4% agarose in MEM containing 2% PBS. After a 5&y incubation monolayers were fixed with 10% formalin and stained with crystal violet. The titer of virus was expressedasplaque-forming units per milliliter (PFU/ml). Interferon assays. The assayfor interferon (IPN) was performed by the cytopathiceffect reduction assay(20). Serial dilutions of the samples to be tested were made in
MEASLES VIRUS IMMUNOSUPPRESSION
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96-well microtiter plates. In parallel, dilutions of an IFN standard were also made. These were added to WISH cell monolayers and after 24 hr the monolayers were challenged with vesicular stomatitis virus (VSV). The monolayers were then stained as described and the plates were read photometrically using a vertical light path automated spectrophotometer (Dynatech laboratories, VA). Prostuglundin assay. Prostaglandin E (PGE) was measured using the PGE kits from Seragen(Seragen Inc., MA) and by the methods of Rigler et al. (2 1). Preparation of peripheral blood mononuclear cell suspensions. Peripheral venous blood was drawn from normal individuals in preservative-free heparin. PBMC were separated by centrifugation on Ficoll-Hypaque gradients and washed three times with phosphate-buffered saline (PBS). Viability by trypan blue exclusion was always greater than 98%. Peripheral blood lymphocyte subsets. To obtain lymphocyte suspensions depleted of glass-adherent cells, the PBMC were passedthrough a glasswool column (22). The number of monocytes remaining in the preparation was determined using a benzidine dihydrochloride stain (23). T cells and non-T cells were separated by E-rosette formation with S-(2-amino ethyl) isothiouronium bromide (AET)-treated sheep erythrocytes as previously described (24). When rerosetted, the T-cell population always contained fewer than 1% contaminating cells. The non-T-cell population contained fewer than 5% contaminating E-rosette-forming cells. Supernutuntfluid production. PBMC, 0.8 X 106/ml, were incubated for 24 hr in 2.0 &ml phytohemagglutinin (PHA). For some experiments the PHA was omitted. The supernatant fluid (SN) was then discarded and the cells were infected with measlesvirus or mock infected. After a 72-hr incubation, the cells were pelleted by centrifugation and the SN was collected. The SN was ultracentrifuged at 152,OOOgfor 2 hr and assayed for infectivity. SN always contained less than 10 PFU/ml of measlesvirus. Infected cell cultures. The infected cell pellets from which the SN fluids were collected were then resuspended in RPM1 1640 and the PBMC were irradiated with 3300 rad using a 13’Cssource (Isomedix, Inc., NJ). This dosagerendered the PBMC incapable of significant thymidine uptake while retaining cell viability. After irradiation the PBMC were resuspended in fresh medium. Lymphocyte proliferation assays. All PBMC were cultured in RPM1 1640 medium supplemented with 10% FBS and penicillin and streptomycin. An optimal amount of PHA (2.0 pg/ml) was added. PBMC (1 X 105)were added to each well of a 96-well microtiter plate. Depending on the experiment, dilutions of either the viral suspension (or uninfected control) or the supematant fluids or the infected, irradiated cells were added. The total volume of each well was brought to 250 ~1. Plates were incubated for 66 hr, pulsed with 2.0 X IO-’ Ci of [3H]thymidine, and harvested 12-18 hr later on glasswool filters on a Skatron cell harvester (Skatron, Inc., VA). RESULTS The efict of measles virus on PHA-stimulated lymphocytes. Initially, experiments were carried out to establish the parameters by which measles virus inhibited PHAstimulated proliferation of lymphocytes. Figure 1 shows the concentration of measles virus necessaryto inhibit lymphoproliferation. At a multiplicity of infection (moi) of
370
SANCHEZ-LANIER ET AL. 70
F @
50
f-
40
“0 F
30
g
20
P g
60
10
I I
c 0.001
+ 0.01
MULTIPLICITY
I
I
0.1
1.0
I 10
OF INFECTION (MOI)
FIG. 1.Measlesvirus-inducedinhibition of lymphoproliferation.Measlesvirus, at increasingmultiplicitiesof infection, wasaddedto PBMC which werestimulatedwith PHA (2.0&nl). The percentageinhibition wasdeterminedby comparingthe meancpm of virus-treatedculturesto the meancpm of culturesto which correspondingdosesof control tluid wereadded.Eachpoint representsthe meanpercentageinhibition f SD for four experiments.Control uninfectedfluids did not causeinhibition.
1.Oor greater measlesvirus significantly (P < 0.000 1 by the Student &test) inhibited the proliferation of PBMC. Below an moi of 0.1 the inhibition became negligible. PBMC treated with Vero control fluids, that is, mock infected, did not causesignificant inhibition (data not shown). When suboptimal (0.5 &ml) concentrations of PHA were used to stimulate the PBMC, the percentageinhibition causedby measlesvirus was either higher than the inhibition obtained with optimal doses of PHA or it remained the same (data not shown). The inhibition of lymphoproliferation by measlesvirus did not merely alter the kinetics af the lymphocyte responseto PHA (Fig. 2). The PBMC were stimulated with PHA and infected with measlesvirus at an moi of 1.0, or mock infected with the Vero control, Cultures were then pulsed and harvestedevery 24 hr for the life of the culture. At a harvest time of 24 hr postinfection, the proliferation of the PBMC was too low to be measuredby the uptake of [3H~ymidine. By 48 hr postinfection, there was significant uptake of the [3HJthymidine and someinhibition of the proliferation of the infected PBMC. At 72,96, and 120hr postinfection there was significant (P < 0.001 by the Student t test) inhibition by measlesvirus of lymphoproliferation. Despite the inhibition observed,cell viability was always greater than 85% and in most caseswasgreaterthan 95%. The e$ct of infected cells on PHA-stimulated lymphocytes. PHA-stimulated lymphocyteswere infected with measlesvirus or mock infected for 72 hr. The cells were then irradiated with 3200 rad which wasshown to prevent further proliferatian while retaining cell viability and added to fresh PHA-stimulated autologousPBMC at varying ratios of infected, irradiated cells to fresh cells The resulting proliferation of the freshPBMC is shown in Fig. 3. Inhibition causedby the measlesvirus-infected,irradiated lymphocytes of the proliferative responseof the fresh PBMC was highly significant (P K O.,OOl)at ratios ranging from 1:1 to 1:128 infected PBMC to fresh PBMC. The greatestamount of inhibition was seenwhen there was equal numbers of fresh lymphocytes ( 1 X 105)and irradiated lymphocytes ( 1 X 10s)in eachwell ( f : 1). How-
MEASLES VIRUS IMMUNOSUPPRESSION
16 TIME OF CULTURE
371
120
HARVEST
(hours after infection)
FIG. 2. The kinetics of measles-induced inhibition of lymphoproliferation. Peripheral blood lymphocytes were stimulated with PHA (2.0 &ml) infected with measles virus and pulsed and harvested over a 5-day period. The data are expressedas mean cpm + SD for triplicate cultures from one donor and are representative of three experiments. There was no signi$cant difference in cell viability between control and infected cultures at any time.
64
66
46
16.
6-
FIG. 3. The inhibitory effect of irradiated, measles virus-infected peripheral blood lymphocytes on the response of fresh autologous PBMC to PHA. Lymphocytes which were either mock infected or infected with measles virus and then irradiated with a dose that prevented further proliferation were added in varying numbers to fresh autologous PBMC. The proliferative response of the fresh PBMC to PHA was measured by uptake of [3H]thymidme. Results are expressedas mean f SD for five experiments. (0) Mockinfected PBMCResh PBMC. (0) Measles virus-infected PBMCResh PBMC.
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ET AL.
12345071 Donor moi.o.01
Mv moi.l.0
FIG. 4. The inhibitory effects of measles virus compared to the inhibitory effects of irradiated, infected PBMC on lymphoproliferation. Measles virus at an moi of either 0.01 or 1.Owas added directly to lymphocytes which had been stimulated with PHA. Irradiated, infected lymphocytes were assayed for infectious measlesvirus by plaque assayand added to fresh autologous PBMC at a concentration that established an moi of 0.01. For each donor reported cultures were done in triplicate and the variance was less than 10%. The two bars representing measlesvirus added directly are the four experiments as reported in Fig. 1.
ever, significant inhibition was still observed with some donors when there was only one irradiated, infected lymphocyte for every 512 fresh autologous lymphocytes. Lymphocytes which had been mock infected and irradiated did not affect the proliferation of the fresh autologous PHA-stimulated lymphocytes. The viability of the irradiated cells was greater than 92% for the life of the culture so the inhibition was not due to cell death. The PBMC which had been irradiated were all plaque assayedfor the presence of infectious measlesvirus. The cells were assayedby directly absorbing them to the cell monolayer for 1.5 hr or by sonicating them first for cellular disruption. No appreciable difference was found in the amount of infectious virus using the two different methods. Figure 4 compares the inhibition of PHA-induced proliferation caused by direct addition of measles virus to the inhibition caused by infected, irradiated lymphocytes. The infected, irradiated PBMC which had been assayedfor infectious virus were added in ratios ranging from 1:16 to 1:64, infected to fresh PBMC, such that measlesvirus was added at an moi of 0.0 1. Direct addition of measlesvirus at an moi of 0.0 1 caused substantially less inhibition. Measles virus added at an moi of 1.O caused comparable or slightly less inhibition. Therefore, inhibition caused by the measlesvirus-infected, irradiated PBMC was greater than that caused by direct addition of measles virus and at an moi which was lOO-fold lower than that needed to cause inhibition by direct addition of measlesvirus. Measles virus is known to undergo substantial replication only in PBMC which have been stimulated with PHA. Supematant fluids collected from PBMC which had been infected with measles virus in the absence of PHA contained fewer than 10 PFU/ml. However, if these same PBMC were then stimulated with PHA, even 96 hr postinfection, substantial amounts of virus could be obtained in the SN. Thus it was necessaryto determine whether or not the lymphocytes needed to be stimulated with PHA prior to infection with measlesvirus in order to causeinhibition of lymphoproliferation. Figure 5 shows the results of an experiment where the PBMC were either
MEASLES VIRUS IMMUNOSUPPRESSION
Control
1:4
cl
UNSTWJATED INFECTED IRRMIAITD PBL
o
WA
S”YULA,EC
1:8
1:16
373
INFECTED
1:32
FIG. 5. A comparison of the effect of stimulated and unstimulated measles virus-infected, irradiated PBMC on lymphoproliferation. Lymphocytes were incubated in the presence or absence of PHA for 24 hr. They were then infected with measles virus. The ratios expressed on the abscissa are the ratios of measles-infected, irradiated PBMC to fresh PBMC.
stimulated with PHA or left unstimulated for 24 hr and then either infected with measles virus or mock infected. After 72 hr in the absence of PHA the PBMC were irradiated and added to autologous PBMC to determine whether substantial replication of measles virus in the PBMC prior to irradiation was necessaryto observe the inhibition. All infected PBMC regardless of whether they had been stimulated with PHA prior to infection were inhibitory, although the inhibition causedby the unstimulated infected PBMC was significantly less than that caused by stimulated infected cells. Therefore, it was not necessaryfor the virus to undergo multiple cycles of replication in the lymphocytes prior to the addition of fresh lymphocytes, for inhibition to be observed. In spite of the fact that substantially lessvirus could be cultured from measlesvirusinfected PBMC than the amount of virus needed to suppress lymphoproliferation it was still possible that the measlesvirus and not the cells was suppressing the lymphoproliferation. Therefore, antibody to measles virus was added to cultures of PHAstimulated lymphocytes and to virus-infected irradiated PBMC. Two sources of antimeaslesvirus antibody were used (Table 1). Autologous serum from measles-seropositive donors was shown to partially reverse the inhibition caused by the infected PBMC at the ratios shown in Table 1. However, by plaque titration the autologous serum did not totally neutralize the infectious virus. Higher titered serum from patients with subacute sclerosing panencephalitis (SSPE) was also used. (Serum was a gift from Larry E. Davis, Department of Neurology, University of New Mexico, School of Medicine.) Serum from several patients were used to avoid obtaining results which might be associated with a particular patient. A range of serum dilutions were used so that the concentration of serum varied from that dilution of serum which just neutralized infectious virus (usually a 150 dilution of serum) to an excess of neutralizing antibody. All cultures were checked by plaque titration to assure that all virus was neutralized. From Table 1 it can be seen that the SSPEserum completely reversed the inhibition caused by the irradiated, measles virus-infected cells. The
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TABLE 1 The Effect of Antibody to Measles Virus on the Inhibition of Lymphocyte Proliferation Caused by Measles Virus-Infected Lymphocytes % Inhibition Donor serum*
Infected:uninfected”
No serum
Autologous serum
SSPE
1:6 1:16 1:8 1:16 I:16 1:32
82 72 91 88 79 71
49 38 55 46 ND’ ND
6 18 10 16 1 5
1 2 3
a Measles virus-infected irradiated lymphocytes were added in the indicated ratios to fresh PHA-stimulated lymphocytes. * Sera from patients with subacute sclerosing panencephalitis were added so that all infectious measles virus was effectively neutralized. ’ Not done.
SSPEserum was then absorbedwith measlesvirus-infected Vero cellsand the serum added to cultures of infected irradiated PBMC and PHA-stimulated PBMC. The absorption of the SSPEserum was verified by the elimination of immunofluorescent staining of measles-infectedVero cells.The inhibitory effectsof the cellswere significantly although not completely restored (Table 2). As an additional control, autologous and SSPEserawere added to cultures of PHA-stimulated PBMC to which measlesvirus (moi = 1, 5) was added directly. The SSPEserum completely neutralized the infectious virus and reversedthe inhibitory effectsof the virus. The autologous serum did not completely neutralize the virus and only partially reversedthe inhibitory effectsof the virus (data not shown). TABLE 2 The Effect of Absorbing Out Antibody to Measles Virus from SSPE Serum on the Inhibition of Lymphocyte Proliferation Caused by Measles Virus-Infected Lymphocytes 90Inhibition
Infecteduninfected” 1:4 1:s 1:16
No serum
SSPEserum *
Mock absorbed serumC
Absorbed serumd
91 87 83
6 6 7
8 16 12
42 43 43
0 Measles virus&fected irradiated lymphocytes were added at the indicated ratios to fresh PHA-stimulated lymphocytes. * Serum from patients with subacute sclerosing panencephalitis was added so that all infectious measles virus was effectively neutmlized. c SSPEserum was absorbed using mock-infected Vero celIs. d SSPEserum was absorbed using measlesvirus-infected Vero cells.
MEASLES VIRUS IMMUNOSUPPRESSION
i
375
1
TIME OF CCUECTION OF SUPERNATWTS hJn oner i7fectioil) FIG. 6. The inhibitory effectsof supematant fluids on PHA-induced proliferation of lymphocytes. Supernatant fluids were collected from cultures of lymphocytes at 24-hr intervals following infection with measlesvirus or mock infection. Data are expressed as mean cpm 2 SD for triplicate cultures.
The efict of supernatant fluids on lymphoproliferation. Another possible mechanism for the measlesvirus-induced immunosuppression is the production of suppressive soluble substancesby the measlesvirus-infected lymphocytes. SN were collected from measlesvirus-infected and mock-infected PBMC at 24&r intervals. After ultracentrifugation the SN were assayedfor infectivity and found to contain less than 10 PFU/ml. The virus-free SN were added to fresh PBMC stimulated with PHA at a dilution of I:5 or 1:10. The results of these experiments are shown in Fig. 6. Supematant fluids collected from PBMC immediately following infection with measlesvirus (0 hr) or 24 hr later did not significantly affect the response of PBMC to PHA. The SN collected at later times from measlesvirus-infected PBMC were inhibitory when compared with SN collected from mock-infected controls. Supematant fluids collected from PBMC which had been treated with Vero control material (mock infected) were not generally inhibitory to mitogenesis when compared to the untreated control (data not shown). For all subsequent experiments, SN were collected from measles virus-infected PBMC 72 hr after infecting the cells. The inhibitory potential of the SN collected at this time showed some donor variation. When optimal concentrations of PHA (2.0 &ml) were used, the mean percentage inhibition caused by the SN was 39%. In the cases of suboptimal concentrations of PHA (0.5 &ml), the mean percentage inhibitions was 60%. It was then of interest to determine whether infected PBMC must produce infectious virus in order to produce the suppressive SN. The PBMC were either stimulated with PHA for 24 hr or left unstimulated, then infected with measles virus or mock infected, and the SN were collected after 72 hr as usual. Table 3 compares the effect that SN collected from both stimulated and nonstimuIated PBMC had on lymphoproliferation. Both sets of SN were inhibitory; thus production-of infectious virus partic& was not a prerequisite to produce suppressive SN. Verification of viral infection of stimulated PBMC was made by detectionof virus by plaque assay.
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TABLE 3 The Effect of PHA Stimulation on the Production of Suppressive Supematant Fluids by Measles Virus-Infected Lymphocytes Unstimulated”
Donor 1 2
Vero control 83,726’ (12,876) 40,181 (795)
PHA stimulated*
virus
Vero
virus
infected
% Inhibition
control
infected
% Inhibition
48,373 (2,586) 28,568
42
79,463 (4,390) 38,799
39,019 (2,766) 25,954
51
(2,223)
(1,369)
(2,795)
29
33
’ PBMC were incubated with media for 24 hr prior to either mock infecting or infecting with measles virus. The SN were collected after 72 hr and added to fresh PBMC. b PBMC were stimulated with PHA for 24 hr prior to either mock infecting or infecting with measles virus. The SN were collected after 72 hr and added to fresh PBMC. c Data are expressed as mean cpm (SD) for triplicate cultures.
The role of lymphocytesubsetsin the production of the suppressiveSN. In order to determine which subsets of PBMC might be responsible for producing the suppressive soluble substance various cell subpopulations were assayed.First, the adherent cell population, consisting primarily of monocytes, was removed from the PBMC suspensions by passing the lymphocytes over glasswool columns. The resulting nonadherent cell population (~2% monocytes by histochemical criteria) was stimulated with PHA for 24 hr and then infected with measlesvirus or mock infected. After 72 hr the SN were collected and added to fresh, PHA-stimulated PBMC. Supematant fluids always contained fewer than 10 PFU/ml. There was no difference in the inhibition caused by SN from nonadherent lymphocytes when compared to the inhibition caused by SN from the unfractionated PBMC (data not shown). The lymphocytes were further fractionated into E-rosette-forming cells (T cells) and non-E-rosetteforming cells (non-T cells) by rosette sedimentation with (AET)-treated sheep erythrocytes. Both of these subpopulations of PBMC were stimulated and infected with measlesvirus and the SN collected after 72 hr. From the data presented in Table 4, it is apparent that the suppressive soluble substance can be obtained from both populations of cells and the inhibition was similar to that caused by SN collected from unfractionated, infected PBMC. Lack of a role for prostaglandins and interferon in the SN. Prostaglandins, which are known to suppresslymphoproliferation, might be contained in the SN and cause the suppression. To examine this, indomethacin (1 pg/ml), a prostaglandin synthetase inhibitor, was added to measles virus-infected and mock-infected PBMC and supematant fluids were collected after 72 hr of incubation. The SN which had been collected from PBMC treated with indomethacin were as inhibitory as SN collected when indomethacin was not present, suggestingthat prostaglandins were not responsible for the inhibition caused by the SN (data not shown). More direct evidence that prostaglandins were not involved in the suppression caused by the SN was found by measuring the amount of PGE in the SN. PBMC were stimulated with PHA and either infected with measlesvirus or mock infected. Supematant fluids were collected
377
MEASLES VIRUS IMMUNOSUPPRESSION TABLE 4 A Comparison of the Elfect of Supematam Fluids Collected from E-Rosette-Forming Cells and Non-E-Rosette-Forming Cells on Proliferating PBMC Supematant fluids” Donor
Virus-infected*
I
-
Unfractionated
+
114,703e (3,114) 69,155
-
97,512
+
70,435 (2,224) 81,110 (3,494) (47,325) (1,748)
(2,454) 2
(3,442) 3
-
+
E-RFC’
Non-E-RFCd
116,721 (5,918) 61,982 (326) 82,191 ( 1,204) 57,744
124,284 (3,918) 103,640 (5,222) ND/
78,150 (2,831) 49,888
58,707 ( 1,069) 83,818 ( 1,842) 47,73 1
(602)
(2,225)
(2,055)
’ Each of the three populations of lymphocytes was stimulated with PHA and either mock infected or infected with measlesvirus, and supematant fluids were collected 72 hr later. b PBMC were either mock infected (-) or infected with measlesvirus (+). c E-rosette-forming cells contained fewer than 1%contaminating cells when rerosetted. d Non-E-rosette-forming cells contained fewer than 5% contaminating E-rosette-forming cells. ’ Data are expressed as mean cpm (standard deviation) for triplicate cultures. ‘Experiment not done.
24, 48, and 72 hr postinfection and frozen at -70°C for the prostaglandin assay. Supematant fluids collected from measlesvirus-infected PBMC did not differ significantly from SN collected from the mock-infected lymphocytes in their prostaglandin content. Since viral-induced IFN is known to inhibit lymphoproliferation the amount of IFN contained in the SN collected from infected and mock-infected PBMC was directly measured by biological assay.The concentrations of IFN were donor variable, but in caseswhere the same donor was used several times, that donor was found to be consistent. Most SN from measles virus-infected PBMC ranged from 100 to 500 units/ml of IFN. Anti-EN antibody to either EN-(Y or IFN-r (Interferon Sciences, Inc., NJ) was added to the SN from both measles virus-infected and mock-infected PBMC. At the same time, the IPN antibody was added to known samples of IFN to serve as a control. Both the antibody-treated SN and the antibody-treated IFN-a control were added to PBMC which had been stimulated with PHA and the incorporation of [3H]thymidine was measured. The results of these experiments are shown in Tables 5 and 6. The anti-IFN-a! completely reversed the inhibitory effectsof the IFN(Yon the proliferating PBMC (Table 5). However, the anti-IFN-cl! had no effect on the SN collected from the measles virus-infected PBMC such that, with or without the IFN antibody, the SN was still inhibitory (Table 6). Similar results were obtained when antibody to IFN-7 was added to the SN (data not shown). Thus, antibodies to either IF%(u or IFN-y had no effect on the inhibitory effects of SN. The antibody-
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ET AL.
TABLE 5 The Effect of Interferon-a and Antibody to Interferon-a on Proliferating PBMC IFN--y + antibody to 1FN-y b IPNq (units) Donor
PHA”
500
100
15
1
2.0 0.5 2.0 0.5
31’ 63 30 71
28 57 30 67
18 48
2.0 0.5 2.0 0.5
35 72
35 62 27 37
20 44
2 3 4
ND ND
25 50
10 26
2000 units IPN--y +2OOO+NUAb
500 units 1FN-r + 1OOONUAb
0 0 0 0 NDd ND ND ND
0 0 0 0 ND ND ND ND
@Concentration PI-IA in &ml. ‘Antibody to IFN (neutralizing units/ml) completeIy neutralixed the active IPN-a as measured by plaque reduction assay. ’ Data are expressed as percentage inhibition of media controt d Not done.
TABLE 6 The Effect of Antibody to Interferon-a on Inhibitory Supernatant Fluids Antibody to IFN-o (neutralizing units/mI)“ Donor
PHAb
None
5ooo
2000
loo0
500
1
2.0 0.5 2.0 0.5 2.0 0.5 2.0 0.5 2.0 0.5 2.0 0.5
23” 50 29 54 40 42 20 83 38 64 37 54
NDd ND 25 50 ND ND ND ND ND ND ND ND
22 52 16 31 ND ND <5 69 ND ND ND ND
22 55 23 45 48 40 <5 73 43 65 37 63
18 57 ND ND ND ND <5 74 ND ND ND ND
2 3 4 5 6
a Ail concentrations of antibody to IPN completely neutral&d the active 1PN-u as measured by a plaque reduction assay. bConcentration PI-IA in &ml. ’ Data are expressed as percentage inhibition of media control. dNotdone.
MEASLES VIRUS IMMUNOSUPPRESSION
379
containing SN were also assayedby biological assayfor IFN and there was no detectable IFN in the SN. Finally, inhibitory SN collected from PBMC which had been infected but not prestimulated with PHA contained little detectable IFN. These data would indicate that IFN is not the suppressive agent in the SN. DISCUSSION Even though numerous laboratories have investigated the mechanisms responsible for measles virus-induced suppression, it remains poorly understood. The suppression may be due to a direct effect of the virus on cell proliferation and to a viralinduced change in the function of the infected lymphocyte. This study was designed to investigate the latter possibility. First, it was shown that measlesvirus induced the release of a suppressive soluble factor. The suppression caused by this soluble factor was not due to measlesvirus, PGE, or IFN. Second, production of infectious virus was not required to produce the suppressive soluble factor. Third, measlesvirus-infected lymphocytes were able to suppress lymphocyte proliferation at a concentration of virus which was lOO-fold lower than the concentration of measles virus usually required to induce such suppression; yet this suppressive activity could be blocked by measlesvirus antibody. The literature contains conflicting reports regarding the role of a suppressive soluble factor in measlesvirus-induced immunosuppression. When such a factor has been found, its properties vary with the report. For example, measlesvirus-infected HeLa cells produced a soluble factor suppressive to DNA synthesis (25). The production of that particular soluble factor required infectious virus. Neighbour and Bloom found that SN from measles virus-infected lymphocytes were suppressive to concanavalin A responses.This suppressive activity was attributed to IFN (26). Salmi et al. found that SN collected from measlesvirus-infected lymphocytes were also suppressive (27). In this case,antibodies to IFN abolished the suppression only partially suggestingthat other factors contributed more to the suppression than IFN. Hirsch et al. found a suppressive soluble factor in 9 of 20 SN collected from PBMC of measles patients and cultured in the absence of any stimulant (28). This suppressive substance was not further characterized. In patients with acute measles, the [‘H]thymidine uptake of lymphocytes with either PHA or candida antigen was lower in media containing autologous serum than in media containing FBS (29). This soluble inhibitor also was not further characterized. Two previous reports were unable to find any suppressive activity in SN generated from measlesvirus-infected PBMC ( 17,30). One reason for the differences might be in the Edmonston strain of virus used. Volckaert-Vervliet et al. reported that different strains of measles virus, and even virus of the same strain but from different sources,induced widely variable amounts of IFN (3 1). It is conceivable that this strain variation could apply to other soluble substances in addition to IFN. While the above reports on the presence of a suppressive soluble factor were often conflicting, the present study brings up several interesting points. Similar to the studies of Hirsch et al. some variation in the ability of different donor lymphocytes to produce a suppressive soluble substance was observed. It is conceivable that such variation in the ability to produce a suppressive factor might be extrapolated to differences in patients who get complications of measles. The present report showed
380
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ET AL.
for the first time that measles virus contained within unstimulated PBMC could induce a suppressive soluble factor. Since only a limited number of lymphocytes in the body respond to a measlesinfection with proliferation, and the majority of lymphocytes in the circulation are in a resting stage, this is highly significant. An infected lymphocyte does not need to be proliferating in order for the virus to alter lymphocyte function. This also means that a small number of infected cells producing such suppressive factors can have an effect on other uninfected lymphocytes or even other types of cells. This provides another mechanism by which the virus could aid in its own dissemination and persistence as well as facilitate secondary infections. The present study showed that the amount of infectious virus recovered from sup pressive irradiated cells was significantly lower than the titer of virus required to induce suppression in fresh cells. In spite of this, the suppression still appeared to be due to measles virus since antibody to the virus reversed the suppression. There are several possible explanations. The tirst possibility is that some kind of temperaturesensitive or other type of mutant was developed in culture but not measured by the standard plaque assay.In this case,enough virus would be present to causethe inhibition but it simply was not measured by the assay.Second, the virus could also induce a suppressive soluble factor. The present study did show that such a mechanism is operative during measles infection although it is unclear if the production of such a factor can entirely explain the phenomenon addressed above. A third possibility is that infection of the lymphocytes by the virus altered the properties of the measles virus making it far more suppressive. Such a mechanism might explain why in the case of some viral infections the immunosuppressive effects are high while the amount of virus present is low. Lastly, infection of the fresh, PHA-stimulated lymphocytes may be more efficient when the virus is cell associated. This does not seem likely due to the long replication cycle of the virus, the relatively short term of the culture, and the low ratios of infected cells to uninfected cells. In addition, when measlesvirus is added to PBMC cultures at an moi of 10 so that all cells are infected, the percentage inhibition is never greater than 80% which is lower than the maximum inhibition obtained in this study. In summary, measlesvirus has been shown to inhibit lymphocyte proliferation by the induction of a suppressive soluble factor and by suppressive infected, irradiated cells. The production of such suppressive factors during viral infection may explain some of the profound immunosuppression seen in situations in which little or no infectious virus can be recovered. ACKNOWLEDGMENTS The authors thank Mary Padilla for her help in preparing the manuscript, Stella Falter and Anita Follins for technical assistance,and Michael McChesney, M.D., for his helpful suggestions.
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8. Osunkoya, B. O., Adeleye, G. I., Adejumo, T. A., and Sahmonu, L. S., Arch. Ogesamte Virusforshung 44,323, 1974. 9. Coovadia, H. M., Parent, M. A., Loening, W. E. K., Wesley, A., Burgess, B., HaIlett, F., Brain, P., Grace, J., Naidoo, J., Smythe, P. M., and Vos, G. H., Amer. J. Clin. Nutr. 27,665, 1974. 10. Joffe, M. I., and Rabson, A. R., Clin. Immunol. Immunopathol. 20,2 15, 1981. 11. Joseph, B. S., Lampert, P. W., and Oldstone, M. B. A., J. Virol. 16, 1638. 12. Huddlestone, J. R., Lampert, P. R., and Oldstone, M. B. A., C/in. Zmmunol. Zmmunopathol. 15,502, 1980. 13. Casali, P., Rice, G. P. A., and Oldstone, M. B. A., J. Exp. Med. 159, 1322, 1984. 14. Pelton, B. K., Hylton, W., andDenman, A. M., Clin. Exp. Immunol. 47, 19, 1982. 15. McChesney, M. B., Fujinami, R. S., Lampert, P. W., and Oldstone, M. B. A., J. Exp. Med. 163, 1331, 1986. 16. Galama, J. M. D., Ubels-Postma, J., Vos, A., and Lucas, C. J., Cell. Zmmunol. 50,405, 1980. 17. Sullivan, J. L., Barry, D. W., Albrecht, P., and Lucas, S. J., J. Immunol. 114, 1458, 1975. 18. Lucas, C. J., Galama, J. M. D., and Ubels-Postma, J., Cell. Immunol. 32,70, 1977. 19. Rapp, T., J. Bacterial. 88, 1448, 1964. 20. Yeh, T.-J., McBride, P. T., Overall, J. C., Jr., and Green, J. A., J. Clin. Microbial. 16,4 13, 1982. 21. Rigler, G. L., Peake, G. T., and Ratnee, A., J. Endocrinol. 70,285, 1976. 22. Greaves, M., and Brown, G., J. Immunol. 112,420,1914. 23. Kaplow,L. S., Blood26,215, 1965. 24. Pellegrino, M. A., Ferrone, S., Dierich, M. P., and Reisfeld, R. A., Clin. Immunol. Immunopathol. 3, 324, 1975. 25. Minegawa, T., Nakaya, C., and IbA, H., J. Virol. 13, 118, 1974. 26. Neighbour, P. A.,and Bloom, B. R., Proc. Natl. Acad. Sci. USA 76,476, 1979. 27. Salmi, A., Vydelingum, S., Salonen, R., and Ilonen, J., Fed. Proc. 44,15 18,1987. 28. Hirsch, R. L., Griffin, D. E., Johnson, R. T., Cooper, S. J., DeSoriano, I. L., Roedenbeck, S., and Vaisberg, A., Clin. Immunol. Immunopathol. 31, 1, 1984. 29. Whittle, H. C., Dossetor, J., Oduloju, A., and Bryceson, A. D. M., J. Clin. Invest. 62,678, 1978. 30. Lucas, C. J., Ubels-Postma, J., Galama, J. M. D., and Rezee, A., Cell. Zmmunol. 37,448, 1978. 3 1. Volckaert-Vervliet, G., Heremans, H., de Ley, M., and Billiau, A., J. Gen. Virol. 41,459.