CELLULAR
IMMUNOLOGY
47, 241-241 (1979)
Effects of Purified BURTON ZWEIMAN, Allergy
Measles Virus Components Human Lymphocytes’
ROBERT
P. LEAK,
on Proliferating
DAVID WATERS, AND HILARY
KOPROWSKI
and Immunology Section, Department of Medicine, and the Department of Neurology, University of Pennsylvania School of Medicine and the Wistar Institute, Philadelphia, Pennsylvania 19104 Received August 16, 1978
Preparations of purified measles virus and both core and membrane-rich fractions suppressed mitogen-induced proliferation of human lymphocytes without reducing viability or the number of T lymphocytes in culture. The suppressive activity of the measles virus was heat labile. The virus preparations by themselves did not induce lymphocyte proliferation.
INTRODUCTION The possible role of immune responses to the measles virus in disease has prompted interest in the interaction between the measles virus and human lymphocytes. It has been shown that the measles virus can be grown in cultures of peripheral blood cells (1) or lymphoblastoid lines (2). However, conflicting results have been obtained in attempts to demonstrate in vitro stimulation by measles virus of lymphocytes from putatively measles-immune subjects (3-6). It has been suggested (6) that only certain measles virus preparations are active in this regard, but the viral preparations utilized have been relatively crude. Another factor possibly playing a role is a suppressive effect of measles virus itself on lymphocyte reactivity. Studies in this laboratory (7,8) have shown a dosedependent inhibition by unfractionated measles virus preparations of lymphocyte proliferation without evidence of cytotoxicity, as measured by several parameters. In some studies, there was indirect evidence that neutralization of virus infectivity prevented this suppressive effect. At present it is still unclear whether stimulation and suppression of lymphocytes are functions of different components of the measles virus. We have utilized a highly purified measles virus and fractions thereof, as prepared by our group (9), to help understand measles-lymphocyte interactions. This report describes some of these studies. MATERIALS
AND METHODS
Measles virus purification. Confluent monolayers of AV-3 (human amnion) cells were infected with measles virus (Broxato strain). After 46 hr, the culture medium ’ Supported by the University of Pennsylvania- Wistar Institute Multiple Sclerosis Research Center (5P02-NS 11037)and 5TOl-A10703. 241 000%8749/79/120241-07$02.00/O Copyright 0 1979by AcademicPress,Inc. All rights of reproductionin any form reserved.
242
ZWEIMAN
ET AL.
from the infected cells was removed and clarified of large debris by low-speed centrifugation. The virus-containing medium was then concentrated 10x with an Amicon ultrafiltration apparatus using an XM-300 Diaflow membrane. The concentrated virus-containing medium was centrifuged at 76,SOOgfor 1 hr at 4°C through 30% (w/w) sucrose in 0.01 M Tris, pH 7.4,0.15 M NaCl, 0.001 M EDTA (NTE) buffer onto a 55% (w/w) sucrose NTE cushion. The cloudy 30-55% interface material (whole virus) was collected and diluted 4 x with NTE buffer, layered onto preformed continuous 20-55% sucrose gradients, and centrifuged for 8 hr at 76,500g at 4°C. These gradients were fractionated into l-ml fractions, densities were determined, and selected fractions were pooled together, diluted with NTE buffer, and pelleted at 89,400g at 4°C for 90 min. These pellets VP-2 (1.20- 1.22 g/cc) and N- 1 (1.25- 1.26 g/cc) were resuspended in NTE buffer at the protein concentrations determined (10). Aliquots were taken for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (11) and for examination by electron microscopy. A 1:5 dilution of the measles virus preparation contained 2.8 x lo5 Pfu/ml. Protein concentrations in the N-l and VP-2 fractions were 4.3 and 3.2 mg/ml, respectively. Virus infectivity assay. Serial 10x dilutions were made of each sample in MEM without calf serum. Each dilution was assayed in duplicate, with 0.5 ml being inoculated per 60-mm petri dish of AV-3 cells. Following 3 hr of incubation at 37°C the inoculum was removed, and the cells were washed with MEM without calf serum and overlayed with carboxymethyl cellulose medium, pH 8.1 (12), containing 5% fetal calf serum. The dishes were incubated at 37°C in 5% CO2 to allow for plaque development. After 5 days, the semisolid overlay was removed, the cells were fixed with formalin and stained with crystal violet, and the number of plaques was counted. Lymphocyte donors and culture technique. Heparinized (10 U/ml) blood was obtained from 35 normal subjects, all with a history of previous measles infection. Mononuclear cells were separated on a Ficoll-Hypaque gradient, with resultant yields averaging 80-95% lymphocytes, 5-20% monocytes (as indicated by phagocytic capacity for latex particles), and occasional granulocytes. Viability, estimated by trypan blue exclusion, was uniformly >97%. Cells were washed twice and resuspended in a final concentration of 5 x lo5 lymphocytes/ml in spinner-modified minimal essential medium (GIBCO) with added glutamine, penicillin-streptomycin, and 10% fetal calf serum (Microbiologic Associates). To half of the replicate cultures in 12 x loo-mm glass vials was added phytohemagglutinin P (Difco) in a final concentration of 10 pg/ml; the other half of the replicates received additional medium instead of PHA. To replicate cultures with and without PHA was also added either (a) a suspension of the whole measles virus in several dilutions; (b) one of the fractions of the measles virus as described above; or (c) a supernatant of the cell line culture used for propagation of the measles virus. The lymphocytes were then cultured for 72 hr at 37°C in 5% CO,/air with a terminal 4-hr pulse of 1.0 @i of tritiated thymidine (specific activity-6.7 CilmM; New England Nuclear Co.). Uptake of isotope into TCA-precipitable material was determined by scintillation spectrometry using methods previously described by us (13) and expressed as counts per minute. Quenching was checked for by external standardization. In some experiments, the effect of the measles virus on T and non-T lymphocytes in the culture population was investigated through a combination of lymphocyte
PURIFIED
MEASLES
VIRUS
EFFECTS
ON LYMPHOCYTES
243
rosetting and autoradiography. Lymphocytes were cultured with and without PHA and measles virus fractions as described above. After 72 hr including a terminal 4-hr pulse of tritiated thymidine, cells from replicate culture vials were pooled, pelleted, and washed twice with Hank’s solution. The cells were suspended in a concentration of 1.O x 106/mland rosetted with sheep erythrocytes (E rosettes) for detection of T lymphocytes as previously utilized in our laboratory (14). The resuspended cells were then gently layered onto well-cleaned glass slides, dried, fixed, and coated with NTB-2 tracking emulsion for autoradiography as previously described by us (15). Emulsion-coated slides were incubated in the dark at 4°C for 1 week, then developed, fixed, and stained. A cell was considered labeled when the nucleus contained at least 5 times the number of grains seen in a similarly sized background area. In this way, the percentage of proliferating cells in the presence or absence of PHA stimulation was enumerated, and the percentage of those cells which were T and non-T lymphocytes could also be simultaneously determined. RESULTS The mean incorporation of tritiated thymidine into lymphocytes of 10 subjects cultured with either (a) PHA, (b) the whole Broxato measles virus preparation, or (c) the N-l (nuclear core) and VP-2 (membrane-rich) fractions is shown in comparison with the control cultures (Table 1). Both unheated and heated (56°C for 1 hr) measles virus preparations were assayed for culture periods of both 3 and 7 days. PHA-induced proliferation was measured at 3 days. No evidence of proliferation was seen with any of the measles virus preparations in either the 3-day or the 7-day cultures. If anything, there was a measurable (though not statistically significant) decrease in lymphocyte isotope incorporation in the presence of unheated measles virus preparations. The effects of the addition of the measles virus or its fractions to lymphocytes incubated with a strong stimulating concentration of PHA are shown in Table 2. The results using lymphocytes of 25 subjects are expressed in summary form here. Because of the considerable variation in the degree of PHA-induced isotope incorporation among lymphocytes of individual subjects, the effect of added virus is expressed as an inhibition ratio (+SEM): mean cpm in cultures with PHA and measles virus fraction mean cpm in cultures with PHA alone The mean isotope incorporation was significantly lower (P < 0.01) in the PHAincubated cultures containing unheated preparations of the whole measles virus, and of both the N-l and the VP-2 fractions, in a dose-dependent fashion when compared with cells incubated with PHA alone. By comparison, the whole virus and N-l fractions which had previously been heated at 56°C did not significantly suppress PHA-induced responses. A moderate degree of suppression was seen in the cultures containing heated VP-2. In four of the experiments selected at random, the infective measles virus concentration was determined in the supematant fluid at the end of the culture period (Table 2). Infective measles virus was consistently present in those cultures to which unheated N-l and VP-2 had been added. In control experiments equal aliquots of these unheated measles virus fractions were
244
ZWEIMAN
ET AL.
TABLE 1 Lymphocyte Stimulation by PHA or Measles Virus Isotope incorporation” PHAb Whole measles virus” Unheated Heated N- 1 fraction (1:SO) Unheated Heated N-l fraction (1500) Unheated Heated VP-2 fraction (150) Unheated Heated VP-2 fraction (1:500) Unheated Heated Control culture supernatec
45,500 * 5,100 375 + 250 550 k 350 350 2 200 600 k 300 300 + 150 475 k 250 470 + 300 670 k 310 510 r 240 705 k 275 500 + 275
a Mean cpm incorporation of tritrated t SEM after 7 days of culture-triplicate determination in each of 10 subjects. * See text for protein concentrations and infectivity of viral preparations. r Supernatant fluid from monolayer culture used for virus propagation without added virus.
cultured in the same medium, but without lymphocytes, at 37°C for a period of 72 hr. At that time no infective measles virus was found in the medium. Also no infective virus was found in culture fluids to which the heated N-l or VP-2 fraction or PHA alone had previously been added. Infective virus was seen in the supematant of one of the four cultures containing heated VP-2. The mean viability of lymphocytes at the end of the culture period in 12 experiments is also shown in Table 2. The mean percentage of cells excluding trypan blue at the end of the culture period was lower in those cultures containing all of the measles preparations, both heated and unheated. However, there was no correlation between the mean degrees of viral-induced inhibition and the effects of the viral preparation on lymphocyte viability. In six experiments (Table 3), PHA-incubated lymphocytes cultured with or without measles virus or virus fractions were analyzed for the percentage of cells rosetting sheep erythrocytes (T lymphocytes), isotope-labeled cells by autoradiography (proliferating lymphocytes), and cells with both properties. The percentage of autoradiographically positive cells was significantly reduced from that seen with PHA incubation alone (32 k 5%) when either the unheated virus or the N-l or VP-2 fraction was also present in the culture (22 +- 4%, 15 2 7%, and 13 f lo%, respectively). By contrast, the percentage of isotope-labeled cells in the cultures containing PHA plus heated virus fractions were not significantly different from those with PHA alone. These findings generally correlate with the findings detected by scintillation counting (Table 2). The suggestion by autoradiographic study that the heated VP-2 fraction itself might stimulate lymphocyte proliferation independ-
245
PURIFIED MEASLES VIRUS EFFECTS ON LYMPHOCYTES TABLE 2
Effects of the Presence of Measles Virus Fractions on PHA-Induced Lymphocyte Proliferations
Measles virus
Inhibition ratio*
Mean viability’
0.55 + 0.10 0.95 2 0.15
68 5 8% 70 k 4
0.33 f 0.12 0.91 f 0.11
60 -c 5 64+-4
4,12,7,8 w40,0,
0.60 I 0.15 0.94 k 0.07
-
22,14,2,4 37,0,0,0 o,o,o,o,
0.27 + 0.08 0.71 + 0.12 -
63 + 5 70 k 4 76 k 4
Infectivity in lymphocyte culture0
Whole virus (1:50) Unheated Heated N-l (1:50) Unheated Heated N-l (1: 100) Unheated Heated VP-2 (1:50) Unheated Heated PHA
(1Concentration of measles virus (in PFU/ml) in lymphocyte culture supernatant fluid after 72 hr (4 experiments). * Inhibition ratio: (mean incorporation of t&rated thymidine-PHA and measles)/(mean incorporation of tritrated thymidine-PHA alone).
ent of the effects of PHA was not confirmed in the scintillation counting studies (Tables 1 and 2). The depression of PHA-induced proliferation by the unheated measles virus preparation did not appear to be due to a reduction in the percentages of T lymphocytes. At the termination of the culture, the mean percentage of E-rosetting cells in those cultures containing PHA plus the various virus fractions was not significantly lower than that with PHA alone (Table 3). It was found that only a portion of the isotope-labeled cells rosetted sheep RBC in the PHA-incubated cultures, similar to that previously observed by us in antigen-stimulated lymphocytes (16). A similar pattern of E rosetting of the labeled cells was seen in the cultures with or without the added measles virus or virus fractions (Table 3). DISCUSSION These studies utilizing a highly purified measles virus obtained from a human infection confirmed results of our earlier experience (7) with a less purified measles vaccine virus preparation. Addition of the measles virus in vitro suppressed mitogen-induced lymphocyte proliferation. Such suppression was not seen following the addition of a heated virus preparation which was no longer infective. Although lymphocyte viability may be somewhat less in cultures containing the added measles virus fractions, there was no significant difference in viability between those cultures in which lymphocyte proliferation was suppressed (with unheated virus) and those in which no such suppression of proliferation occurred (with heated virus). These findings suggest that infectivity, by this viral strain at least, may be required for the suppressive effect. An alternative explanation is that viral particles or fractions bind to the lymphocytes without true infectivity and a
246
ZWEIMAN TABLE
ET AL. 3
Effects of Measles Virus on Proliferating Culture combination PHA alone PHA and unheated measles N-l (1:50) PHA and heated measles N-l (1:50) PHA and unheated measles VP-2 (1:50) PHA and heated VP-2 (150) PHA and whole unheated virus (1:50) PHA and whole heated virus (1:50) PHA and control culture supematantc
E rosettes”
T and Non-T Lymphocytes Labeled cell@ (%I 32?
5
15 t
7
Labeled rosetted cells (%)
62 +
8’
12 + 4
66?
13
76t
6
31 k 10
27 + 6
71 + 12
13 + 10
824
59 k 19
46+
8
58?
3
22+
1
622
4
342
7
11 2 1
64*
6
332
7
14 t 6
6?1
19 k 7 821
(1Cultured lymphocytes binding at least three sheep erythrocyteslcell. b Isotope-labeled cells (*5 times the grain count in background). c Control culture supernatant-supernatant fluid from monolayer culture used for propagating measles virus-but with no virus added.
the
heat-labile component of the virus is responsible for the suppression of lymphocyte reactivity. An additional finding of these studies is that the suppressive effect was exerted by both a highly purified nuclear core fraction and a membrane-rich component of the virus. At the same time, these fractions did not significantly suppress the low levels of spontaneous isotope incorporation seen in cultures without added mitogen. The latter finding suggests that the measles effect was not just a nonspecific suppression of lymphocyte activity. In addition, there was no alteration in the relative numbers or reactive patterns of T lymphocytes. The presence of suppressive activity in the nuclear core material could explain degrees of immunosuppression associated with the postulated “slow virus” infection with defective measles virus (17, 18). In this model, a protective immune response is not elicited to the degree that the virus is eliminated in the manner seen after acute self-limited infections. Yet, as in the case of subacute sclerosing panencephalitis (SSPE), very high levels of antibodies are formed against measles antigens (18, 19). Therefore, it has been postulated by some (20) that the chronic progressive infection in SSPE is related to a suppressed cell-mediated immune response to the measles virus. Studies in this laboratory (21) and elsewhere (22) have shown transient depression of skin test and in vitro lymphocyte reactivities to standard antigens following measles immunization of normal humans. It is not clear whether such suppression seen after in vivo measles virus administration extends to the immune lymphocyte response to the measles virus itself. To answer this question, a measles virus antigen that consistently stimulates immune lymphocytes in vitro is needed. In earlier studies, most investigators had not found that the measles virus preparation used by them would stimulate proliferation of
PURIFIED MEASLES VIRUS EFFECTS ON LYMPHOCYTES
247
lymphocytes from humans with histories of measles infection and demonstrable humoral antimeasles immunity. Graziano, Ruckdeschel, and their colleagues (5,23) reported that a commercially prepared unpurified complement-fixation test measles antigen preparation stimulated lymphocyte proliferation, whereas the control culture medium did not. These authors attributed the stimulatory capacity of their material to the high concentration of membrane-associated antigens. More recent studies by that group (24) and elsewhere (6) have shown a considerable variation in stimulatory capacity among different lots of the same preparation from the same source, presumably prepared by the same techniques. It has been postulated that this variance could be due, at least in part, to mycoplasma contamination of the viral cultures (24). In the studies reported here, we have not been able to demonstrate any in vitro stimulation of the lymphocytes from measles-immune subjects with the highly purified measles virus fractions employed. The serum of a number of these subjects checked at random contained complement-fixing antibodies against the commercially prepared antigens referred to above. In pilot trials by us, additions of this complement-fixing antigen (from two lots) in serial dilutions to lymphocytes of these subjects for cultures of varying duration did not result in cellular stimulation. Use of higher concentrations of our purified virus or the commercial preparation led to toxic effects on the lymphocytes. Therefore, it appears that demonstration of immune lymphocyte reactivity to measles virus by this assay is still a variable finding, possibly related to factors such as the source and purity of the measles preparation. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
Sullivan, J. L., Barry, D. W., Lucas, S. J., and Albrecht, P., J. Exp. Med. 142, 773, 1977. Barry, D. W., Sullivan, J. L., Lucas, S. J., et al., J. Immunol. 116, 89, 1976. Saunders, M., Chambers, M. E., Knowles, M., et al., Lancer 1, 72, 1969. Ahmed, A., Strong, D. M., Sell, K. W., et al., J. Exp. Med. 139, 902, 1974. Graziano, K. D., Ruckdeschel, J. C., and Mardiney, M. R., Cell. Immunol. 15, 347, 1975. Kreeffenberg, J. G., and Loggen, H. G., Cell. Immunol. 33, 443, 1977. Zweiman, B., J. lmmunol. 106, 1154, 1971. Zweiman, B., and Miller, M., Int. Arch. All. Appl. Zmmunol. 46, 822, 1974. Ciangoli, A. K., Lisak, R. P., Zweiman, B., Koprowski, H., and Waters, D., J. Neural. SC;. 28, 33, 1976. 10. Lowry, 0. H., Rosebrough, N. J., Farr, A. J., and Randall, R. J.,J. Biol. Chem. 193,265, 1951. 11. Waters, D. J., and Russell, R. H., Virology 55, 554, 1973. 12. Waters, D. J., Thesis, University of Kansas, 1973. 13. Zweiman, B., Maibach, H., Pappagianis, D., and Hildreth, E. A., J. fmmunol. 102, 1283, 1969. 14. Lisak, R. P., Levinson, A. I., Zweiman, B., and Abdou, N. I., Clin. Exp. Immunol. 22, 30, 1975. 15. Zweiman, B., Int. Arch. All. Appl. Immunol. 43, 600, 1972. 16. Levinson, A. I., Lisak, R. P., Zweiman, B., and Wilkerson, L. D., Immunology 33, 621, 1977. 17. Horta-Barbosa, L., Hamilton, R., Whittig, B., Fuccillo, D. A., and Sever, J. L., Science 173, 840, 1971. 18. Ter Meulen, V., Katz, M., and Muller, D., Curr. Trends Microbial. 57, 1, 1972. 19. Connolly, J. H., Allen, I. V., and Hurwitz, L. J., Lancer 1, 542, 1967. 20. Jabboor, J. T., Roare, J. A., and Sever, J. L., Neurology 19, 929, 1969. 21. Zweiman, B., Papagianis, D., Maibach, H., and Hildreth, E. A., Int. Arch. All Appl. Immunol. 40, 834, 1971. 22. Fireman, P., and Kumate, J., J. Ped. 43, 264, 1969. 23. Ruckdeschel, J. C., Graziano, K. D., and Mardiney, M. R., Cell. Immunol. 17, 11, 1975. 24. Ruckdeschel, J. C. Kramarsky, B., and Mardiney, M. R., Cell Immunol. 20, 110, 1975.
IMMUNOLOGY
47, 241-241 (1979)
Effects of Purified BURTON ZWEIMAN, Allergy
Measles Virus Components Human Lymphocytes’
ROBERT
P. LEAK,
on Proliferating
DAVID WATERS, AND HILARY
KOPROWSKI
and Immunology Section, Department of Medicine, and the Department of Neurology, University of Pennsylvania School of Medicine and the Wistar Institute, Philadelphia, Pennsylvania 19104 Received August 16, 1978
Preparations of purified measles virus and both core and membrane-rich fractions suppressed mitogen-induced proliferation of human lymphocytes without reducing viability or the number of T lymphocytes in culture. The suppressive activity of the measles virus was heat labile. The virus preparations by themselves did not induce lymphocyte proliferation.
INTRODUCTION The possible role of immune responses to the measles virus in disease has prompted interest in the interaction between the measles virus and human lymphocytes. It has been shown that the measles virus can be grown in cultures of peripheral blood cells (1) or lymphoblastoid lines (2). However, conflicting results have been obtained in attempts to demonstrate in vitro stimulation by measles virus of lymphocytes from putatively measles-immune subjects (3-6). It has been suggested (6) that only certain measles virus preparations are active in this regard, but the viral preparations utilized have been relatively crude. Another factor possibly playing a role is a suppressive effect of measles virus itself on lymphocyte reactivity. Studies in this laboratory (7,8) have shown a dosedependent inhibition by unfractionated measles virus preparations of lymphocyte proliferation without evidence of cytotoxicity, as measured by several parameters. In some studies, there was indirect evidence that neutralization of virus infectivity prevented this suppressive effect. At present it is still unclear whether stimulation and suppression of lymphocytes are functions of different components of the measles virus. We have utilized a highly purified measles virus and fractions thereof, as prepared by our group (9), to help understand measles-lymphocyte interactions. This report describes some of these studies. MATERIALS
AND METHODS
Measles virus purification. Confluent monolayers of AV-3 (human amnion) cells were infected with measles virus (Broxato strain). After 46 hr, the culture medium ’ Supported by the University of Pennsylvania- Wistar Institute Multiple Sclerosis Research Center (5P02-NS 11037)and 5TOl-A10703. 241 000%8749/79/120241-07$02.00/O Copyright 0 1979by AcademicPress,Inc. All rights of reproductionin any form reserved.
242
ZWEIMAN
ET AL.
from the infected cells was removed and clarified of large debris by low-speed centrifugation. The virus-containing medium was then concentrated 10x with an Amicon ultrafiltration apparatus using an XM-300 Diaflow membrane. The concentrated virus-containing medium was centrifuged at 76,SOOgfor 1 hr at 4°C through 30% (w/w) sucrose in 0.01 M Tris, pH 7.4,0.15 M NaCl, 0.001 M EDTA (NTE) buffer onto a 55% (w/w) sucrose NTE cushion. The cloudy 30-55% interface material (whole virus) was collected and diluted 4 x with NTE buffer, layered onto preformed continuous 20-55% sucrose gradients, and centrifuged for 8 hr at 76,500g at 4°C. These gradients were fractionated into l-ml fractions, densities were determined, and selected fractions were pooled together, diluted with NTE buffer, and pelleted at 89,400g at 4°C for 90 min. These pellets VP-2 (1.20- 1.22 g/cc) and N- 1 (1.25- 1.26 g/cc) were resuspended in NTE buffer at the protein concentrations determined (10). Aliquots were taken for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (11) and for examination by electron microscopy. A 1:5 dilution of the measles virus preparation contained 2.8 x lo5 Pfu/ml. Protein concentrations in the N-l and VP-2 fractions were 4.3 and 3.2 mg/ml, respectively. Virus infectivity assay. Serial 10x dilutions were made of each sample in MEM without calf serum. Each dilution was assayed in duplicate, with 0.5 ml being inoculated per 60-mm petri dish of AV-3 cells. Following 3 hr of incubation at 37°C the inoculum was removed, and the cells were washed with MEM without calf serum and overlayed with carboxymethyl cellulose medium, pH 8.1 (12), containing 5% fetal calf serum. The dishes were incubated at 37°C in 5% CO2 to allow for plaque development. After 5 days, the semisolid overlay was removed, the cells were fixed with formalin and stained with crystal violet, and the number of plaques was counted. Lymphocyte donors and culture technique. Heparinized (10 U/ml) blood was obtained from 35 normal subjects, all with a history of previous measles infection. Mononuclear cells were separated on a Ficoll-Hypaque gradient, with resultant yields averaging 80-95% lymphocytes, 5-20% monocytes (as indicated by phagocytic capacity for latex particles), and occasional granulocytes. Viability, estimated by trypan blue exclusion, was uniformly >97%. Cells were washed twice and resuspended in a final concentration of 5 x lo5 lymphocytes/ml in spinner-modified minimal essential medium (GIBCO) with added glutamine, penicillin-streptomycin, and 10% fetal calf serum (Microbiologic Associates). To half of the replicate cultures in 12 x loo-mm glass vials was added phytohemagglutinin P (Difco) in a final concentration of 10 pg/ml; the other half of the replicates received additional medium instead of PHA. To replicate cultures with and without PHA was also added either (a) a suspension of the whole measles virus in several dilutions; (b) one of the fractions of the measles virus as described above; or (c) a supernatant of the cell line culture used for propagation of the measles virus. The lymphocytes were then cultured for 72 hr at 37°C in 5% CO,/air with a terminal 4-hr pulse of 1.0 @i of tritiated thymidine (specific activity-6.7 CilmM; New England Nuclear Co.). Uptake of isotope into TCA-precipitable material was determined by scintillation spectrometry using methods previously described by us (13) and expressed as counts per minute. Quenching was checked for by external standardization. In some experiments, the effect of the measles virus on T and non-T lymphocytes in the culture population was investigated through a combination of lymphocyte
PURIFIED
MEASLES
VIRUS
EFFECTS
ON LYMPHOCYTES
243
rosetting and autoradiography. Lymphocytes were cultured with and without PHA and measles virus fractions as described above. After 72 hr including a terminal 4-hr pulse of tritiated thymidine, cells from replicate culture vials were pooled, pelleted, and washed twice with Hank’s solution. The cells were suspended in a concentration of 1.O x 106/mland rosetted with sheep erythrocytes (E rosettes) for detection of T lymphocytes as previously utilized in our laboratory (14). The resuspended cells were then gently layered onto well-cleaned glass slides, dried, fixed, and coated with NTB-2 tracking emulsion for autoradiography as previously described by us (15). Emulsion-coated slides were incubated in the dark at 4°C for 1 week, then developed, fixed, and stained. A cell was considered labeled when the nucleus contained at least 5 times the number of grains seen in a similarly sized background area. In this way, the percentage of proliferating cells in the presence or absence of PHA stimulation was enumerated, and the percentage of those cells which were T and non-T lymphocytes could also be simultaneously determined. RESULTS The mean incorporation of tritiated thymidine into lymphocytes of 10 subjects cultured with either (a) PHA, (b) the whole Broxato measles virus preparation, or (c) the N-l (nuclear core) and VP-2 (membrane-rich) fractions is shown in comparison with the control cultures (Table 1). Both unheated and heated (56°C for 1 hr) measles virus preparations were assayed for culture periods of both 3 and 7 days. PHA-induced proliferation was measured at 3 days. No evidence of proliferation was seen with any of the measles virus preparations in either the 3-day or the 7-day cultures. If anything, there was a measurable (though not statistically significant) decrease in lymphocyte isotope incorporation in the presence of unheated measles virus preparations. The effects of the addition of the measles virus or its fractions to lymphocytes incubated with a strong stimulating concentration of PHA are shown in Table 2. The results using lymphocytes of 25 subjects are expressed in summary form here. Because of the considerable variation in the degree of PHA-induced isotope incorporation among lymphocytes of individual subjects, the effect of added virus is expressed as an inhibition ratio (+SEM): mean cpm in cultures with PHA and measles virus fraction mean cpm in cultures with PHA alone The mean isotope incorporation was significantly lower (P < 0.01) in the PHAincubated cultures containing unheated preparations of the whole measles virus, and of both the N-l and the VP-2 fractions, in a dose-dependent fashion when compared with cells incubated with PHA alone. By comparison, the whole virus and N-l fractions which had previously been heated at 56°C did not significantly suppress PHA-induced responses. A moderate degree of suppression was seen in the cultures containing heated VP-2. In four of the experiments selected at random, the infective measles virus concentration was determined in the supematant fluid at the end of the culture period (Table 2). Infective measles virus was consistently present in those cultures to which unheated N-l and VP-2 had been added. In control experiments equal aliquots of these unheated measles virus fractions were
244
ZWEIMAN
ET AL.
TABLE 1 Lymphocyte Stimulation by PHA or Measles Virus Isotope incorporation” PHAb Whole measles virus” Unheated Heated N- 1 fraction (1:SO) Unheated Heated N-l fraction (1500) Unheated Heated VP-2 fraction (150) Unheated Heated VP-2 fraction (1:500) Unheated Heated Control culture supernatec
45,500 * 5,100 375 + 250 550 k 350 350 2 200 600 k 300 300 + 150 475 k 250 470 + 300 670 k 310 510 r 240 705 k 275 500 + 275
a Mean cpm incorporation of tritrated t SEM after 7 days of culture-triplicate determination in each of 10 subjects. * See text for protein concentrations and infectivity of viral preparations. r Supernatant fluid from monolayer culture used for virus propagation without added virus.
cultured in the same medium, but without lymphocytes, at 37°C for a period of 72 hr. At that time no infective measles virus was found in the medium. Also no infective virus was found in culture fluids to which the heated N-l or VP-2 fraction or PHA alone had previously been added. Infective virus was seen in the supematant of one of the four cultures containing heated VP-2. The mean viability of lymphocytes at the end of the culture period in 12 experiments is also shown in Table 2. The mean percentage of cells excluding trypan blue at the end of the culture period was lower in those cultures containing all of the measles preparations, both heated and unheated. However, there was no correlation between the mean degrees of viral-induced inhibition and the effects of the viral preparation on lymphocyte viability. In six experiments (Table 3), PHA-incubated lymphocytes cultured with or without measles virus or virus fractions were analyzed for the percentage of cells rosetting sheep erythrocytes (T lymphocytes), isotope-labeled cells by autoradiography (proliferating lymphocytes), and cells with both properties. The percentage of autoradiographically positive cells was significantly reduced from that seen with PHA incubation alone (32 k 5%) when either the unheated virus or the N-l or VP-2 fraction was also present in the culture (22 +- 4%, 15 2 7%, and 13 f lo%, respectively). By contrast, the percentage of isotope-labeled cells in the cultures containing PHA plus heated virus fractions were not significantly different from those with PHA alone. These findings generally correlate with the findings detected by scintillation counting (Table 2). The suggestion by autoradiographic study that the heated VP-2 fraction itself might stimulate lymphocyte proliferation independ-
245
PURIFIED MEASLES VIRUS EFFECTS ON LYMPHOCYTES TABLE 2
Effects of the Presence of Measles Virus Fractions on PHA-Induced Lymphocyte Proliferations
Measles virus
Inhibition ratio*
Mean viability’
0.55 + 0.10 0.95 2 0.15
68 5 8% 70 k 4
0.33 f 0.12 0.91 f 0.11
60 -c 5 64+-4
4,12,7,8 w40,0,
0.60 I 0.15 0.94 k 0.07
-
22,14,2,4 37,0,0,0 o,o,o,o,
0.27 + 0.08 0.71 + 0.12 -
63 + 5 70 k 4 76 k 4
Infectivity in lymphocyte culture0
Whole virus (1:50) Unheated Heated N-l (1:50) Unheated Heated N-l (1: 100) Unheated Heated VP-2 (1:50) Unheated Heated PHA
(1Concentration of measles virus (in PFU/ml) in lymphocyte culture supernatant fluid after 72 hr (4 experiments). * Inhibition ratio: (mean incorporation of t&rated thymidine-PHA and measles)/(mean incorporation of tritrated thymidine-PHA alone).
ent of the effects of PHA was not confirmed in the scintillation counting studies (Tables 1 and 2). The depression of PHA-induced proliferation by the unheated measles virus preparation did not appear to be due to a reduction in the percentages of T lymphocytes. At the termination of the culture, the mean percentage of E-rosetting cells in those cultures containing PHA plus the various virus fractions was not significantly lower than that with PHA alone (Table 3). It was found that only a portion of the isotope-labeled cells rosetted sheep RBC in the PHA-incubated cultures, similar to that previously observed by us in antigen-stimulated lymphocytes (16). A similar pattern of E rosetting of the labeled cells was seen in the cultures with or without the added measles virus or virus fractions (Table 3). DISCUSSION These studies utilizing a highly purified measles virus obtained from a human infection confirmed results of our earlier experience (7) with a less purified measles vaccine virus preparation. Addition of the measles virus in vitro suppressed mitogen-induced lymphocyte proliferation. Such suppression was not seen following the addition of a heated virus preparation which was no longer infective. Although lymphocyte viability may be somewhat less in cultures containing the added measles virus fractions, there was no significant difference in viability between those cultures in which lymphocyte proliferation was suppressed (with unheated virus) and those in which no such suppression of proliferation occurred (with heated virus). These findings suggest that infectivity, by this viral strain at least, may be required for the suppressive effect. An alternative explanation is that viral particles or fractions bind to the lymphocytes without true infectivity and a
246
ZWEIMAN TABLE
ET AL. 3
Effects of Measles Virus on Proliferating Culture combination PHA alone PHA and unheated measles N-l (1:50) PHA and heated measles N-l (1:50) PHA and unheated measles VP-2 (1:50) PHA and heated VP-2 (150) PHA and whole unheated virus (1:50) PHA and whole heated virus (1:50) PHA and control culture supematantc
E rosettes”
T and Non-T Lymphocytes Labeled cell@ (%I 32?
5
15 t
7
Labeled rosetted cells (%)
62 +
8’
12 + 4
66?
13
76t
6
31 k 10
27 + 6
71 + 12
13 + 10
824
59 k 19
46+
8
58?
3
22+
1
622
4
342
7
11 2 1
64*
6
332
7
14 t 6
6?1
19 k 7 821
(1Cultured lymphocytes binding at least three sheep erythrocyteslcell. b Isotope-labeled cells (*5 times the grain count in background). c Control culture supernatant-supernatant fluid from monolayer culture used for propagating measles virus-but with no virus added.
the
heat-labile component of the virus is responsible for the suppression of lymphocyte reactivity. An additional finding of these studies is that the suppressive effect was exerted by both a highly purified nuclear core fraction and a membrane-rich component of the virus. At the same time, these fractions did not significantly suppress the low levels of spontaneous isotope incorporation seen in cultures without added mitogen. The latter finding suggests that the measles effect was not just a nonspecific suppression of lymphocyte activity. In addition, there was no alteration in the relative numbers or reactive patterns of T lymphocytes. The presence of suppressive activity in the nuclear core material could explain degrees of immunosuppression associated with the postulated “slow virus” infection with defective measles virus (17, 18). In this model, a protective immune response is not elicited to the degree that the virus is eliminated in the manner seen after acute self-limited infections. Yet, as in the case of subacute sclerosing panencephalitis (SSPE), very high levels of antibodies are formed against measles antigens (18, 19). Therefore, it has been postulated by some (20) that the chronic progressive infection in SSPE is related to a suppressed cell-mediated immune response to the measles virus. Studies in this laboratory (21) and elsewhere (22) have shown transient depression of skin test and in vitro lymphocyte reactivities to standard antigens following measles immunization of normal humans. It is not clear whether such suppression seen after in vivo measles virus administration extends to the immune lymphocyte response to the measles virus itself. To answer this question, a measles virus antigen that consistently stimulates immune lymphocytes in vitro is needed. In earlier studies, most investigators had not found that the measles virus preparation used by them would stimulate proliferation of
PURIFIED MEASLES VIRUS EFFECTS ON LYMPHOCYTES
247
lymphocytes from humans with histories of measles infection and demonstrable humoral antimeasles immunity. Graziano, Ruckdeschel, and their colleagues (5,23) reported that a commercially prepared unpurified complement-fixation test measles antigen preparation stimulated lymphocyte proliferation, whereas the control culture medium did not. These authors attributed the stimulatory capacity of their material to the high concentration of membrane-associated antigens. More recent studies by that group (24) and elsewhere (6) have shown a considerable variation in stimulatory capacity among different lots of the same preparation from the same source, presumably prepared by the same techniques. It has been postulated that this variance could be due, at least in part, to mycoplasma contamination of the viral cultures (24). In the studies reported here, we have not been able to demonstrate any in vitro stimulation of the lymphocytes from measles-immune subjects with the highly purified measles virus fractions employed. The serum of a number of these subjects checked at random contained complement-fixing antibodies against the commercially prepared antigens referred to above. In pilot trials by us, additions of this complement-fixing antigen (from two lots) in serial dilutions to lymphocytes of these subjects for cultures of varying duration did not result in cellular stimulation. Use of higher concentrations of our purified virus or the commercial preparation led to toxic effects on the lymphocytes. Therefore, it appears that demonstration of immune lymphocyte reactivity to measles virus by this assay is still a variable finding, possibly related to factors such as the source and purity of the measles preparation. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
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