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Cellular immune response to measles mumps and vaccinia viruses in multiple sclerosis.

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Cellular Immune Response
to Measles, Mumps, and Vaccinia Viruses
in Multiple Sclerosis
Henry F. McFarland, MD, and Dale E. McFarlin, M D
The cell-mediated immune response to measles, mumps, and vaccinia viruses was studied in MS patients, normal
controls, and neurological disease controls using a lymphocyte proliferation assay. A small but significant difference was found in the response to measles between the MS and normal control groups but not between the MS
and neurological disease control groups. In each of the groups, the response of both peripheral blood leukocytes
and purified T-cells to measles was significantly less than the response to mumps or vaccinia. The lower response to
measles was not due to the presence of blocking factors or to lack of antigenicity of the measles virus preparation
used in the assay. These findings suggest that differences exist in the normal immune response between these
viruses. Precise quantitation of the immunological response to viruses i n MS and other disease states will depend on
identification of the various functionally reactive cell populations.
McFarland HF, McFarlin DE: Cellular immune response to measles, mumps, and vaccinia viruses in multiple
sclerosis. Ann Neurol 6:lOl-106, 1979
Although the cause of multiple sclerosis (MS) remains unknown, measles virus as well as other viral
agents have been implicated as possible causative
factors in the disease, based primarily on the demonstration of elevated serum [ 11 and, more importantly,
cerebrospinal fluid [3, 101 antibody levels to measles
virus among the majority of MS patients. Elevation of
CSF antibody levels t o other viruses including
mumps, rubella, vaccinia, and herpes has also been
reported in MS [7, 111. These findings support the
supposition that MS may be due directly to a persistent infection. Alternatively, the disease may be related to an aberrant immune response to virus, o r the
elevated antibody levels may represent an epiphenomenon not directly related to the cause of
The purpose of this study was to reexamine the
cellular response t o measles virus and, for comparison, the responses t o mumps and vaccinia viruses
in a group of clinically well defined patients and appropriate controls. Only patients with a clinical
classification of definite MS were studied. Further,
patients with severe disability were excluded. To
minimize differences in antigen preparations and t o
standardize the presentation of antigens, a lymphocyte transformation assay employing virus-infected
cell monolayers was used. In addition, separation of
peripheral blood lymphocytes (PBL) into T- and Blymphocytes was performed, and the reactivity of
these cell populations was assessed for each virus.
MS.
Materials and Methods
Additional evidence for a relationship between
measles virus and MS has been derived from the suggestion that the cellular immune response to measles
virus is altered in MS [ 141. This finding, however, has
been inconsistent, and studies using similar or different techniques have failed to detect a significant difference in the cellular immune response to measles
virus between patients and controls [4-6,8]. Further,
it has recently been suggested that the diminished
cellular response to measles virus in MS is related
to the greater disability of patients as compared to
controls [ 131.
The cell-mediated immune response to measles, mumps,
and vaccinia viruses was studied in 2 0 MS patients, 24
healthy controls, and 12 patients with neurological disease
considered nondemyelinating. Only patients who had a
diagnosis of clinically definite MS according to the criteria
of McAlpine "91 were studied. All patients were ambulatory, were between the ages of 20 and 41 years, and were
considered to be in either remission o r a progressive phase
of disease. No patients were studied during an acute exaczrbation or while receiving steroids. Healthy controls were
between the ages of 19 and 43 years and consisted of individuals without a history of serious medical or neurological
disease. Neurological disease controls consisted of indi-
From the Neuroimmunology Branch, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, MD.
Accepted for publication Feb 13, 1979.
Address reprint requests to D~ McFarland, ~~~i~~~~~
chief, NIB,
IRP, NINCDS, Bldg 36, Room 5D-12, Bethesda, MD 20014.
101
viduals with a well-established neurological diagnosis, including the following specific diseases (in 2 patients each):
amyotrophic lateral sclerosis, polymyositis, Parkinson's disease, Huntington's disease, peripheral neuropathy, and
idiopathic seizures. Blood samples were obtained on multiple occasions during the course of the study. Except for
experiments requiring fresh cells for cell separation, lymphocytes obtained from separate blood samplcs were frozen and employed in at least duplicate assays performed at
different times.
Lymphocyte Preparation
Venous blood was drawn in preservative-free heparin (Abbott). Lymphocytes were obtained by density gradient
centrifugation using Ficoll-diatrizoate (LSM solution,
Bionetics). Lymphocytes were used either fresh or frozen
in 7.5% dimethyl sulfoxide in a programmable cell freezer
(Cryomed) and were stored in liquid nitrogen vapor until
used. Lymphocytes were washed twice in Dulbecco's
phosphate-buffered saline (PBS) and adjusted to the appropriate viable cell concentration in RPMI 1640 with
1OOJ A B serum and 1% penicillin and streptomycin. In
some experiments designed to detect blocking factors,
A B serum was replaced by autologous, horse, or rabbit
serum. Enriched populations of T - and B-cells were prepared either by using a sephadex anti-Fab immunoabsorbant column [ 121 o r by rosetting with sheep red blood cells
(SRBC). Two milliliters of 5% SRBC were incubated with
2 ml of neuraminidase (10 unitsiml, Sigma) for 30 minutes.
The SRBC were washed and diluted to a 1% suspension.
Two milliliters of lymphocytes (1 x 107/ml)were mixed
with 2 ml of 1% SRBC suspension in polypropylene
round-bottom tubes, incubated for 30 minutes at 37"C,
spun for 5 minutes at 5 0 0 g , and incubated for an additional
30 minutes at 4°C. T h e pellets were resuspended, and the
rosetted cells were separated from the nonrosetted cells on
a Ficoll-diatrizoate gradient centrifuged for 20 minutes at
500 g. T-cells prepared by either method contained less
than 3% surface Ig-positive cells as detected by staining
with fluorescein-conjugated sheep FAB, antihuman IgM.
The B-cell fraction contained 6 5 to 75% cells which were
positive for surface IgM. B-cell fractions examined for their
ability to form SRBC rosettes contained less than 5%
rosetting cells. No attempt was made to deplete adherent
cells.
Lymphocyte Proliferation Assay
Monolayers of Vero cells were grown in 96-well microtiter
plates (Falcon) and infected with either the Edmonston
strain of measles virus, the Enders strain of mumps virus, o r
the I H D strain of vaccinia virus. Additional wells remained
uninfected. Each well was infected at a multiplicity of approximately 1. Infected wells showed substantial cytopathic
effect by 48 hours after infection. In preliminary studies,
fluorescent antibody staining of parallel monolayers grown
on glass coverslips showed diffuse viral antigen present
at this time. T h e monolayers were fixed with 0.025%
glutaraldehyde for 30 minutes at 4°C and washed three
times with PBS. Lymphocytes were added to each group of
infected and noninfected wells in triplicate at a cell concentration of 3 x lo5 cells per well. The plates were incu-
102 Annals of Neurology Vol 6 No 2
August 1979
bated for 96 hours at 37°C in a 10'9%carbon dioxide atmosphere. Each well was labeled with 1 p C i of tritiated
thymidine for four hours before harvesting in a MASH I1
(Multi Automated Sample Harvester, Microbiological Associates). The filter-paper discs were counted in a liquid
scintillation counter (Beckman). T h e arithmetic mean of
each group of triplicate wells was determined. Stimulation indices for each group were calculated by dividing
the mean counts per minute incorporated o n infected
monolayers by the counts on uninfected monolayers. In
studies comparing patient and control responses, each individual was tested in multiple assays using frozen lymphocytes obtained from a single bleeding. In most instances,
less than a 10% difference existed between separate assays.
The mean response for each individual was calculated from
values obtained in the repetitive assays. Statistical analysis
was performed using the Student t test.
Antibody Binding Assuy
Vero cell monolayers either uninfected o r infected with
measles virus were fixed with 0.025% glutaraldehyde as
just described. Twenty microliters of normal human serum
known to be seropositive for measles virus was added to
triplicate wells at fivefold dilutions. T h e plates were incubated for 30 minutes, washed three times, and incubated
with 20 pl of radioiodinated Staph Protein A (SPA) o r
sheep antihuman IgG for 30 minutes. The wells were
washed three times and air-dried, and the individual wells
were counted in an automatic gamma counter. Additional
assays employed measles virus hyperimmune mouse serum
prepared by repeated inoculation of Balb/c mice with
purified Edmonston strain measles virus in complete
Freund's adjuvant. IgG binding was detected by subsequent incubation with '251-labeled SPA on infected and
noninfected wells. Binding ratios were calculated as the
counts per minute bound on infected monolayers divided
by those bound on noninfected monolayers. A binding
ratio of greater than 2 was considered significant.
Results
The results of the studies on cell-mediated immune
response to measles, mumps, and vaccinia viruses are
shown in Table 1. The difference in mean response
to measles virus between the MS and normal control
groups is significant at the 0.05 level. In distinction,
no significant difference was found between the MS
and neurological disease control groups. The mean
response to mumps and vaccinia viruses did not differ
significantly among any of the groups. However,
clear differences were seen when the magnitude of
the response elicited by the three viruses was compared. In both control groups and the MS group, the
mean response to measles virus was significantly less
than that for either mumps or vaccinia virus (p <
0.00 1). These findings indicate that the cellular response was consistently less for measles virus than for
mumps or vaccinia virus as measured by the lymphocyte proliferation assay employed in this study.
In contrast to the relatively consistent low re-
change in the pattern of their response. All individuals included in the data presented in Table 1 and the
Figure had a positive history for measles and mumps
virus infections and smallpox vaccination. T w o individuals omitted from the Figure who had not been
vaccinated for smallpox had stimulation indices of
less than 2 to vaccinia. T h e 3 individuals in the Figure
who had stimulation indices less than 2 to vaccinia
had, by history, been vaccinated. No responses to
any of the viruses appeared to correlate with age, and
none of the individuals tested had a known history of
reexposure to mumps virus o r recent revaccination
against smallpox.
I n order to assess the low level of lymphocyte
reactivity to measles virus, additional variables of this
assay were examined. First, the possibility that
measles virus antigens were altered o r absent from
the infected monolayers was considered. Measles
virus-infected monolayers prepared identically to
those used in the lymphocyte transformation assay
were employed in a solid-phase radioimmunoassay
using radiolabeled SPA and sheep antihuman I g G
(Table 2). These preparations were found to be exquisitely sensitive for detection of antimeasles antibody in human scrum. Similarly, titers approximately
tenfold greater than neutralization titers were obtained with mouse hyperimmune measles serum
using infected monolayers and iodinated SPA. Thus,
measles antigens were adequately expressed o n the
infected monolayers.
Second, since occasional individuals showed a response to measles that was substantially above the
mean, we examined the possibility that the hypore-
Table I . Lymphocyte Proliferatice Response
of Peripherul Blood L3rnphocyte.r to Measle..r,
hfunips. and Vaccinia Viruse.ra
Mean Stimulation Indexb
~~
Group
No.
Measles
Tested Virus
Mumps
Virus
Vaccinia
Virus
Healthy
24
3.2
8.6 f 5.4
12.5 i 7.8
20
2.2' I 0.8
7.9
12
2.4 :t 1.7
7.5 t 5.8
_t
2.2
control
Multiple
-t
4.7
11.0
%
4.2
sclerosis
Neurological
disease
7.5 t 4.9
control
"Values given are mean stimulation index t SD.
"Stimulation index is the quotient of the incorporation (in cpm) of
tritiated thymidine o n infected monolayers divided by the incorporation (in cpm) o n uninfected monolayers. T h e arithmetic mean
of repetitive assays done o n individuals in each group was used to
calculate the mean for that group.
sponse to measles virus, considerable variation was
found in the responses to mumps and vaccinia viruses
(Figure). This variation does not appear related to t h e
status of the individual when tested. Repetitive testing of 5 individuals (2 controls, 3 patients) at various
times over a two-year period showed n o substantial
MUMPS
MEASLES
VACCINIA
-
10
F
..
.......
a6:
4
-
.....*!I
..........
-It :
;
.-
0'
CONTROLS
.....
.----.
.....
....... 1
..
;
I
MS
OND
CONTROLS
MS
OND
CONTROLS
MS
OND
McFarland and McFarlin: Cellular Immunity in MS
103
Table 2. Binding
(I!
Nornul Human and M o u e Hyperimmune Measles Sera t o Measles-infected Monolayers"
Agent
Used
Dilution
Measles
Infected
Noninfected
Binding
Ratio
1:10
1:50
1:250
1 : 1,250
1:6,2 5 0
1:10
1:50
1:250
1 : 1,250
1:6,250
2 1,485
14,183
4,383
1,389
53 1
4,775
4,145
2,223
581
283
3,378
1,421
49 3
344
316
806
495
277
224
22 5
6.4
10.0
8.9
4.0
1.7
5.9
8.4
8.0
2.6
1.3
10-
15,381
4,804
1,166
600
5,992
2,777
1,200
5 94
5,635
1,492
823
652
5,060
2,221
896
557
2.7
3.2
1.4
0.9
1.2
1.2
1.3
1.1
HUMAN SERUM
'";'I-Staph
protein A
i251-Antihuman 1gG
MOIJSE SERUM
Hyperimmune
measles serum
]0 - 2
10-3
10-4
Normal serum
lo-'
10-2
10-4
aValues given are mean binding (in cpm) on triplicate wells. Human serum was obtained from healthy control sera positive for measles virus
antibody (complement fixation, 1 : 32; hemagglutination inhibition, 1 : 16). Mouse serum was obtained from mice repeatedly immunized
with the Edmonston strain of measles virus (complement fixation, 1: 16). Binding ratio is the ratio of binding o n infected monolayers
versus noninfected monolayers; a binding ratio of 2 or greater is considered significant.
Table 3. Comparison of-the Lymphocyte Prolzferative Response Obtained Using Various Sources of Mammalian Sera'
Uninfected
Serum
Fxperiment 1
1 % AB
15; H o r s e ' '
1 4 Rabbit'
Experimcnt 2
10% AB
1 OC: Autolorrou\
M e a s l e s Virus
M u m p s Virus
Vaccinia Virus
Vero Cells
1,992 (1 0)
2,293 ( 1 6)
1,937 (1 6)
31,952 (15.7)
9,275 (6.4)
17,382 (14 6)
60,818 (29.9)
37,279 (25.6)
64,192 (53.8)
2,037
1,457
1,194
2,976 (1 8)
2.581 (0 9)
49,002 (29 6)
46.904 (16.6)
76,223 (46 1)
87.904 (11 0)
1,654
2.833
Binding ratios are given in parentheses.
"Lymphocytes from a normal donor were prepared in serum-free media and resuspended in media containing the various sera. Values given
are mean of triplicate wells and are expressed as counts per minute (stimulation index).
"The horse serum had no demonstrable neutralizing activity against measles, mumps, or vaccinia virus.
'Serum was ohtaned from young 13 to 4 kg), nonimmunized rabbits.
sponsiveness of some individuals was related to a
serum blocking factor. No differences were found
between autologous and AB serum in the response to
measles, mumps, or vaccinia virus (Table 3). More
important, similar levels of tritiated thymidine incorporation were obtained when human serum was
replaced by horse or rabbit serum lacking antimeasles
activity. It is therefore unlikely that the modest response to measles virus was due to serum blocking
factors .
104 A n n a l s of N e u r o l o g y
Vol 6 No 2 A u g u s t 1079
T h e cellular response to these viruses was further
examined in a group of patients and controls with
respect to the characteristics of the responding cell
populations. Unfractionated PBL and fractionated
populations of T- and B-cells separated either by
affinity column or by SRBC rosetting were tested in
the lymphocyte transformation assay. The results are
shown in Table 4. T h e response to measles and vaccinia viruses was limited to the T-cell population.
T-cells were also the predominant responding cell
Table 4. Comparison of Reactivity
of T- and B-Lymphocytes"
Mean Stimulation
Index
Group
Assayed
Cells
Control
PBL
(N=5) T
B
MS
PBL
(N = 6) T
B
Measles
Virus
Mumps
Virus
1.0 10.1 t 1.9
3.1 8.6 t 3.9
1.2 t 0.5 2.7 2 2.0
2.9 t 1.6 11.8 t 8.9
3.2 t 1.6 6.6 5 5.8
1.0 ? 0.4 5.9 t 5.2
2.1
2
4.5
?
Vaccinia
Virus
18.6 t 7.9
15.0 t 4.8
1.5 t 0.4
16.8 t 5.3
17.4 t 6.6
1.2 t 0.2
aLymphocytes were separated either by using an immunoabsorbant column consisting of purified goat IgG antihuman Fab
coupled to sepharose or by rosette formation with SRBC. T-cell
preparations contained less than 3% surface Ig-positive cells; Bcells varied between 50 and 70% surface Ig-positive cells.
population to mumps virus, although a lesser but
significant B-cell response was often found. A similar
B-cell response was not observed on measles- or
vaccinia-infected monolayers. It seems unlikely that
the response t o mumps found in the B-cell population was due entirely to contaminating T-cells since
the T-cell response was usually greater to vaccinia
than to mumps virus.
The magnitude of the response to each of the viruses studied, expressed as stimulation indices, was
frequently increased with the T-cell population
compared to the unfractionated PBL. In some instances this was partly due to the lower background
obtained with T-cells on uninfected Vero cells.
However, in each of the individuals tested in this
manner, the actual response in counts per minute was
greater with the T-cell fraction than with the unfractionated PBL.
Discussion
The cell-mediated immune response to measles,
mumps, and vaccinia viruses was studied in a population of MS patients, healthy controls, and neurological disease controls employing a lymphocyte proliferation assay. The studies demonstrated a small
difference in response to measles virus between the
MS and normal control groups. It seems unlikely,
however, that this difference is specific for MS since
no difference was found between the MS and neurological disease groups. Although MS patients with
severe disability were excluded from this study, the
findings are consistent with the possibility that the
difference found between the MS patients and the
healthy control group is due to the effect of chronic
disease, as has been previously suggested [ 131. Similarly, the response to mumps and vaccinia viruses
tended to be greater in the healthy control group,
although the mean values were not significantly dif-
ferent. These findings essentially agree with other
recent studies which, using a variety of techniques,
have failed to demonstrate a significant difference in
the cellular response to measles or other viruses between MS patients and controls 14-6, 81.
A marked difference was demonstrated in both the
patient and control groups between the response to
measles virus and that to mumps or vaccinia virus; in
each group, the response to measles virus was
significantly less than that to the other two agents.
This observation indicates that the normal cellular
immune response to measles virus is probably less
than that to mumps or vaccinia viruses. It is uncertain
if this difference is unique to the particular assay
used in our study. Although antibody and lymphocytes may recognize different measles virus determinants, the findings that antigen can be readily demonstrated by antibody and that occasional individuals
have a cellular response well above the mean indicate
that the hyporesponsiveness is not attributable to
lack of viral antigen. A second consideration is that
the low response to measles virus is due to an immune regulatory component. No evidence was found
for a blocking factor in human serum since a similar
pattern of responses was obtained using sera from
other species, but the possibility of a cellular suppressor mechanism specific to measles virus cannot
be eliminated.
Separation of PBL into T- and B-cell populations
indicated that the responses to measles and vaccinia
viruses are essentially limited to the T-cell fraction.
The mumps response is also found predominantly in
the T-cell population, although in some individuals a
small but significant B-cell response occurs. It is unlikely that this B-cell response is solely due to T-cell
contamination because a similar B-cell response was
not observed with vaccinia virus. Although the response of enriched T-cells to measles virus is usually
slightly augmented over that of PBL, the magnitude
of the increase seems most consistent with enrichment of a responding cell population and not loss of
active suppression.
The T-cell population in experimental animals and
humans has been shown to be composed of various
subpopulations which have different functional
properties, including amplification of the immune response, cytolysis or killing of antigen-bearing target
cells, and suppression of humoral and cellular immune responses. Further, the response of these subpopulations is related to the mode of antigen presentation. Compatibility in portions of the major
histocompatibility region is required for lysis of
target cells by killer T-cells [ 151. In distinction, suppressor T-cells have been shown in some systems not
to require histocompatibility in antigen presentation
[2]. Because of these differing requirements among
McFarland and McFarlin: Cellular Immunity in MS
105
T-cell subsets, many biological assays used to investigate human T-cell reactivity map assess only a particular subset of cells.
O u r inability to demonstrate a substantial proliferative response t o measles virus may have resulted
from absence of measles-sensitive cells capable of responding in a proliferative assay o r from inadequate
antigen presentation. T h e response obtained with
mumps and vaccinia viruses could reflect a subpopulation of cells, such as suppressor T-cells, which do
n o t require histocompatibility in antigen presentation t o proliferate. In this case the corresponding
subpopulation of cells sensitized t o measles virus is
diminished.
Although the mechanisms responsible for t h e differences in T-cell responsiveness t o the three common viruses evaluated here are unknown, it is
tempting t o speculate that they may be related to
various biological properties of the viruses. Most importantly, o u r findings indicate that adequate comn the normal cellular immune response to meas1c.s virus and that occurring in specific
diseases such as MS must await clearer definition of
the boundaries of the normal response. Thus, comparison will depend o n the technical ability t o functionally assess the role of T-cells in amplification and
suppression of both cellular and humoral responses
t o measles virus.
2. Broder S, Poplack D, Whang-Peng J , et al: Characterization of
a suppressor-cell leukemia. Evidence for the requirement of
an interaction of two T cells in the development of human
suppressor effector cells. N Engl J M e J 298:66-72. 1978
3. Brody J A , Sever JL, Edgar A, e t al: Measles antibody titers of
multiple sclerosis patients and their siblings. Neurology
(Minneap) 22:492-199, 1972
4. Ciongoli A K , Platz P, D u P o n t B, e t al: Lack of antigen rcsponse t o myxoviruses in multiple sclerosis. Lancet I : 1 I ?',
1972
5 . Cunningham-Rundles S, D uPont B, Posner JB, et al: Lymphocyte transformation in vitro to paramyxovirus antigens in
multiple sclerosis. Lancet 2: 1204, 1975
6. Fuccillo D A , Abela JE, Tra ub R G , et al: Cellular immunity i n
multiple sclerosis. Lancet 1:980, 1975
7 . Kempe CH, Takagayashi K, Miyamato H, e t al: Elevated cerebrospinal fluid vaccinia antibodies in multipltr sclerosis.
Arch Neurol 28:Z78-279, 1973
8. Knowles M, Saunders M: Lymphocyte stimulation with
measles antigen in multiple sclerosis. Neurology (Minneap)
20:700-702, 1970
9. McAlpine D: In McAlpine D , Lumsden CE, Acheson E D
(eds): Multiple Sclerosis: A Reappraisal. Second edition.
Edinburgh and London, (:hurchill/Livingstone, 1972. pp 8 I307
1 0 . Norrhy E, Link 13, Olsson JE: Measles virus antibodies in
multiple sclerosis. Arch N e urol 30:285-292, 1974
1 1 . Norrby E, Link H, Olsson JE, et al: Comparison of antibodies
against different viruses in cerebrospinal fluid and serum s m ples from patients with multiple sclerosis. Infect Immunol
Supportctl in part I>\ National Multiple Sclerosis Society Grant
measles virus in multiple sclerosis: correlation with disability.
Neurology (Minneap) 28:109-112, 1978
14. Utermohlen V, Zabriskie JB: A suppression o f cellular immunity in patients with multiple sclerosis. J Exp Med
028-c-6.
The authors thank Ms L y n n Pedone f o r skillful technical assistailLC.
References
I. Ad a msJM .
Imaga\\a DT: Measles antibodies in multiple sclerosis. Pro< So< t x p Hi01 Mccl I 1 l:562-560, 1962
106 Annals of Neurology
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