Measles virus-polypeptide specificity of the cytotoxic T-lymphocyte response in multiple sclerosis

•Io,m'nalofNtna'oimmunolosy,

F.If.evier

21 (1989)205-212

205

JNI 00690

Measles virus-polypeptide specificity of the cytotoxic T-lymphocyte response in multiple sclerosis Suhayl D h i b J a l b u t , Dale E. McFarl/n and Henry F. McFarland Neuroimmunolo~ Branch, NINCDS, National Institutes of Health, Pethesda, M D 20892, US.A.

(Received16 M'-y1988) (Revised,received19 July1988) (Accepted 19July 1988)

K¢9, words:

Multiplesclerosis;Measlesviruspolypeptide;CytotogicT lymphocyte

summary Som¢ patients with mull/pie sclerosi~ (MS) have been shown previously to have a reduced capacity to generate measles virus (MV)-specific cytotoxic T-lymphocytes (CTLs). The mechanism of this reduction is not understood. Possibilities include sequestration of MV-CTLs within the central nervous system (CNS), abnormalities in regulation of this response (e.g., suppression), a defect in the T-eell repertoire of MS patients and a defect in *he induction or maintenance of the CTL response to MV. To examine these possibilities, the CTL response to three purified polypeptides of MV (hemagglutinin (HA), fusion (F), and nucleocapsid (NC)) was studied in eight healthy controls and 14 patients wigh multiple sclerosis. A defect in the response to two polypeptides of the virus (HA and NC) was found in the MS patien~z with reduced MV-CTL response. The response to F was also reduced but to a lesser e~,tent. Limiting dilution analysis of the MV polypeptide-specific CTL response indicated that suppression is an unlikely cause for the reduction in CTL activity. The lymphoproliferative response to MV, ttA, F, anti NC was comparable in three MS patients and three controls examined. Together, the results of these studies indicate that the reduced MV-CTL response in MS patients was not due to a defect in the T-cell repertoire or sequestration due to cross-reactivity with a single myelin antigen. More likely mec.h~nis.,nsinclude abnormalities in the induction or maintenar.ee of the MV-CTL response or sequestration within the CNS due to recognition of MV antigens.

Introduction Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) of uq-

Address for correspondence:Dr. S. Dhib-Jalbut,NeuroimmunologyB,.anch, Bldg. 10, Rm. 5B-16,NationalInstitute~,of Health, Ikthes~a,MD 20892,U.S.A.

known etiology, l~pidemiological and family studies suggest that both an environmental factor, possibly an infectious agent, and genetic factors contribute to the pathogenesis of this disease (Goodman and McFarfin, 1987). A vital etiology for MS has been proposed based on the demonstration of elevated levels o~" antibodies to several viruses in the spinal fluid of MS patients (Adams and Imagawa, 1962; Albrecht et al., 1983) and the

206

occurrence of primary demyelination in certain naturally occurring (Brooks and Walker, 1984; Haase, 1986; Johnson et al., 1984) and experimental viral infections 0Veiner, 1973; Lipton and Del Canto, 1976). Measles virus (MV) has been linked to the pathogenesis of MS based on serological evidence and the demonstration of an impairment in the generation of MV-specific cytotoxic Tlymphocytes (CTL) in a substantial number of patients with MS (Jacobson et al., 1985). This defect in the generation of MV-CTLs is not due to a reduction in the population of CIM + cells which represent a major component of MV-specific CTLs (Jacobson et al., 1984), but rather due to a reduction in the precursor frequency of MV-CTLs in MS patients as compared to healthy individuals (McFarland et al., 1987). The mechanism responsible for the reduction in MV-CTLs in MS patients is not understood. Possibilities include a defect in the T-ceil repertoire, sequestration of MV-CTLs within the CNS, or abnormalities in the induction or regulation of the CTL response to MY. In healthy individuals MV-specific CI'Ls can be generated in response to at least four MV polypeptides including hemagglutinin (HA), fusion (F), nucleocapsid (NC) and matr;.x protein (Jacobson et al., 1987). In MS, the polypeptidespecific components of the M v - c r L response have not been defined. A reduction in MV-CTL due to a defect in the T cell repertoire or to sequestration of l~fv'-CTLs recognizing a cross-reacting myelin antigen should involve a defect in the response to a single component of MV. In contrast, a defect in the generation or maintenance of immunity to MV or sequestration due to recognition of MV antigens in the CNS should involve a reduction in the response to all or most of the MV polypeptides. In order to investigr.te these possible mechanisms for the reduction in MV-CTL response in MS, the generation of CTLs in response to MV, HA, F, and NC was studied in MS patients and controls. Material and methods

Patients and controls Fourteen patients with definite multiple ~ierosis were studied. Because an objective of this study

was to assess the polypeptide specificity of MVCTL response, MS patients who demonstrate.d, using bulk culture assays, a measurable MV-CTL response were selected. The MV-CTL response in those patients was either moderately reduced or normal in comparison to healthy controls. The control groups consisted of eight healthy individuals whose MV-CTL response range matched with that of the MS patients. All patients and controls had antibodies to MV in their sera indicating prior exposure to the virus.

Purification of MV polypeptides MV polypeptides (HA, F, m~d NC) were purified from a lysate of Daudi cells infected with the Edmonston strain of MY. Purification was done by affinity chromatography using monoclonal antibodies specific to each MV polypepfide coupled to cyanogen bromide-activated Sepharose 4B as described (Bellini et al., 1981). The purity of the MV polypeptides was confiruled by sodium dodecyl sulfate~polyacrylamide electrophoresis (SDS-PAGE) and by enzyme-linked immunosorbent assay (ELISA) as described (Rose et al., 1984). The purified polypeptides appeared on SDS-PAGE as single bands with the appropriate molecular weight and reacted only with the monoclonal antibc'ty specific to a particular polypeptide.

Lymphocyte preparation Peripheral blood lymphocytes (PBLs) obtained from patients and controls by leukophoresis were purified by Ficoll-Hypaque density gradient centrifugation. PBLs were frozen in cryoprotective media (12-1332 A, MA Bioptoducts, Waik.ersville, MD, U.S.A.) using a programmable cell freezer (Cryomedics, Bridgeport, CT, U.S.A.) and were stored in liquid nitrogen vapor until used.

Lymphoproliferative assay PBLs were cultured in a 96-well microtiter plate in RPMI-1640 (Gibco Laboratories, Grand Island, NY, U.S.A.) containing 5~ human AB serum, 15 penicillin/streptomycin, glutamine and Hepes buffer. Feeders (PBLs irradiated with 5000 R) were incubated with media or antigen (MV, HA, F or NC) for 90 min at 37 o C and added to the responder cells in hexaplicate wells. Each well

207 c o n t a i n e d 2 x l 0 s r e s p o n d e r cell, 1 x l 0 s feeder cells, a n d either m e d i u m o r antigen in a final v o l u m e o f 200/~1. T h e c o n c e n t r a t i o n o f M V was 1 x l 0 s p l a q u e - f o r m i n g units ( p f u ) / w e l l a n d t h a t o f t h e purified M V p o l y p e p t i d e s was 1 p g / w e l l . Cultures were c a r d e d for 5 d a y s in a 5 ~ C O 2 h u m i d i f i e d incubator. Cultures were t h e n pulsed w i t h [3H]thymidine (1 / t C i / w e l l ) for 4 h a n d h a r v e s t e d o n a M a s h - l l cell h a r v e s t ~ . T h e lymphoprofiferative r e s p o n s e ( L P R ) was expressed in c p m o f [ s ! ~ t h y m i d i n e u p t a k e a n d as a stimulation i n d e x representing t h e ratio o f [3H]thymidine uptake in stimulated to u n s t i m u l a t e d cells.

targets were t h e n a d d e d to t h e effec~r:, at a c o n c e n t r a t i o n o f 10 ~ cells p e r well. T h e plates were t h e n s p u n (200 r p m for 3 rain) a n d i n c u b a t e d for 4 h in a 5 ~ C O 2 hnmidifu~d incubator. M e a n p e r c e n t lysis was calculated as: ((SlCr.release with sensitized effectors) - ( s p o n t a n e o u s release)) × 1 0 0 / ( ( d e ( e r g e n t r e l e a s e ) - ( s p o n t a n e o u s release)). Antigen-specific lysis was calculated as t h e difference in m e a n lysis b e t w e e n antigen-stimulated a n d u n s t i m u l a t e d effectors cultured w i t h t h e M V infected" target.

Results

C T L assay T h e effector cells w e r e cultured as dczcribed for t h e lymphoproliferative assay except t h a t 12 n d cro-well cultures p e r antigen were p r e p a r e d a n d cultures were carried for 7 days. M V - i n f e c t e d o r u n i n f e c t e d Epstein-Barr virus ( E B V ) - t r a n s f o r m e d autologous B-cells w e r e u s e d as targets. O n d a y 7, targets were c h r o m a t e d for 90 rain at 3 7 ° C in 0.4 m l o f C T L m e d i u m ( R P M I c o n t a i n i n g S~$ fetal c~alf serum) and w a s h e d twice w i t h the s a m e m e d i u m . M V - i n f e c t e d o r u n i n f e c t e d SlCr-laheled

Lymphoproliferative responses Initially, the i m m u n o g e n i c i t y o f t h e purified M V p o l y p e p t i d e s w a s tested b y l y m p h o p r o l i f e r a tion. A n L P R to M V , H A , F, a n d N C was obtained in the three M S p a t i e n t s a n d three c o n t r o l s tested (Table 1). W h e n expressed as c p m (([SH]t h y n ~ d i n e u p t a k e in s t i m u l a t e d ) - (uptake in unstimulated cells)) the r e s p o n s e to all M V antigens was higher in the M S patients t h a n in controls. However, w h e n the responses were expressed as

TABLE 1 LYMPHOPROLIFERATIVE RESPONSES TO MV PEPT1DES IN MS AND CONTROLS

Group Cage No.

[SH]Thymidine uptake 4- standard deviation Media MV HA

Controls 0134

593 + 420

0860

663 4-192

3328

880 4- 277

Mean 4- SD

712 + 150

MS patients 108

2 447 4- 876

111

3410 + 675

113

2508 4- 996

Mean 4- SD

2788 4- 539

• Stimulation it dex.

F

NC

6297 + 3474 (10.6) " 5 272 4- 2 800 (8.0) 14810 + 2709. (16.8) 8 793 + 5 236 (11.8 4- 4.5)

8551 + 1745 (14.4) 10 ~31 4-13 560 (15.8) 15125 4- 3477 (17.2) 1] 402 :.~ 3 372 (15.8 + 1.4)

1500 + 1323 (2.5) 10 284 4- 1851 (15.5) 25 547 + 6800 (29) 12 444 + 12168 (12.3 + 6.7)

4322 4-1 461 (7.3) 6 467 4-1997 (9.8) 19548 4- 8763 (22.3) !0146 + S 299 (13.1 4- 8)

8 357 ± 3 360 (3.4) 29 568 4-13 498 (8.7) 22734 + 5234 (9.1) 20220 4-10826 (7.0 4- 3.2)

12 467 + 2 206 (5.1) 18 203 + 2165 (5.3) 19642 + 5573 (7.8) 16771 4- 3796 {6.1 4-1 5)

14 594 4- 2 421 (6) 16 887 4- 2150 (4.9) 29024 + 7241 (11.6) 20168 4- 7754 (7.5 ~ 3.6)

9 505 ± 3 265 (3.9) 19 369 4- 4 229 (5.7) 12982 + 2 112 (5.2) 17060 4- 6757 (4.9 + 0.9)

208 stimulation indexes, MS patients appeared to have a lower response than the controls. This was due to the higher [3Hlthymidine uptake by the unstimulated cells in the MS patients and is not artifaetu~ since experiments for the MS patients a n d controls were done simuhaneously. Also, increased [3H]thymidine uptake by unstimulated lymphoeytes from MS patients has been observed by others (Reunanen, 1982; Kreuzfelder et al., 1988) and could be due to the presence of activated T-cells. The LPR was not skewed towards a specific MV protein in either the MS patients or controls.

M V - C T L response As discussed above, MS patients who in preliminary studies demonstrated the capacity to generate a moderately reduced or normal MV-CTL response were studied. A t a concentration of 2 x 105 responder cells per well, MV-specific lysis r s n s e d between 4.2 a n d 31.87O with a m e a n of 14.8 4-9.4 in the 14 MS patients compared to a range of 5.5-38.5% in the eight controls with a m e a n of 17.2 + 11.37O. M e a n percent lysis of uninfected targets by M'V-stimulated effectors was 6.0 4- 4.3 in the MS patients a n d 2.3 4- 2.6 in the controls. The MS patients could b e arbitrarily divided into two groups based o n their ability to generate a n M V - C T L response. The first group h~d a relatively normal CTL response (>~ 15%; m e a n 23.8 + 6.57O). The second group h a d reduced b u t measurable C T L response ( < 157O; mean 7.9 + 3.17o). The CTL r e s ~ n s e s to MV, HA, F, and N C for the controls and MS patients with normal MVC T L (response. ~ 157O) are shown in Table 2. Variations in the responses to the M V polypeptides were observed a m o n g individuals in b o t h groups. In general, the C T L response to MV did not appear to b e consistently skewed towards a specific MV protein. In the controls the mean responses to H A and F tended to b e higher than that for NC, a n d in the MS normal responder~ the CTL responses tended to b e higher for F than either H A or N C but the differences did not reach statistical significance. The CTL responses to MV, HA, F and N C in the controls and MS low responders (response

TABLE 2 PERCENT MEASLES VIRUS-SPECIFIC LYSIS IN INDIVIDUALS WITH NORMAL MV-CTL RESPONSES Group Case No.

Antigen MV

HA

Controls 3874 3360 2695 Means±SD

24.7 29.8 38.5 31 +6.9

13.7 17.4 16.7 25.0 21.1 "4.7 27.9 13.7 7.1 22.2+7.5 17.4+ 3.7 12.84-5.1

MS patients 110 113 121 105 137 155 Means+SD

15.2 9.3 24.9 4.5 24.6 5.3 31.8 21.5 29.4 24.3 17.3 5.3 23.8+6.5 11.7+8.9

F

11.5 7.8 16.5 30.7 25.6 1.6 15.6+10.9

NC

10.9 4.9 2.3 26.6 18.2 4.4 11.24-9.5

< 157O) are shown in Table 3. I n the low-responder controls a n d MS patients the C T L response was reduced to all M V proteins tested, b u t in tile M S patients the reduced response for the F-protein was less than for the other peptides.

TABLE 3 PERCENT MEASLES VIRUS-SPECIFIC LYSIS IN INDIVIDUALS WITH LOW MV-CTL RESPONSES Group Case No.

Antigen MV

HA

F

NC

2650 14.8 0134 5.5 0860 13.5 3328 12.9 2434 7.8 4261 7.5 Mean+SD 10 -I-3.8

2.1 3.0 23.4 12.4 0.0 5.9 7.8+8.8

8.4 3.0 27.3 16.2 0.0 2.7 9.6:t:10.4

6.1 8.0 16.2 13.1 0.0 2.8 7.7+6.1

MS patients 119 8.5 108 10.0 111 12.8 150 4.8 125 4.2 112 7.8 102 10.6 103 5.2 Mean+ SD 7.9-1-3.1

0.0 4.9 0.0 0.0 7.4 3.1 2.7 7.1 3.2+3.1

13.2 5.2 0.0 6.6 10.6 6.1 4.9 6.6 6.7+ 3.9

3.1 0.0 7.2 0.0 3.9 0.3 4.1 3.8 2.8+2.5

Controls

209

Comparisons of mean specific iysis in the low and normal responders for each MV protein are shown in Fig. 1. When mean specific lysis for each polypeptide was compared in the MS low responders to that in rite MS normal responder$, the responses were significantly lower for H A ( P = 0.025) and N C ( P = 0.032). The difference in the response to F approached statistical significance ( P = 0.052). The mean CTL response to each MV polypeptide in the MS low responders was compared to that in the control low responders. The response was reduced in the MS group to HA, F, and NC; however, only the reduction in the response t o N C was statistically significant ( P <

NR.CONTROLS IIm LR-CONTROLS 40,

~ u

[]

NR-MS

~1

LR-MS

3o

!,°

O.O5).

0

MV

HA

F

NO

Limiting dilution analysis

ANTIGEN

Fig. 1. Measlesvires-specificlysi~in MS patients and controls. NIt, normal respondets; LR, low responders. MV

25

In six Iow-responder MS patients and five lowresponder controls the CYL response to MV polyHA

25

15 1

(J

g

o

2

' 5

-

-

'

'

I

OI

F

LU

¢~ 20 .=

4

4

2

1

N u m b e r o f ceils x 1 0 5 / m i c r o t i t e r well

Fig. 2. Measles virus antigen-specificCTL responses at decreasing responder cell concentrations. Closed circles represent mean lysis for fivecontrols; open circlesrepresent mean lysis for six MS patients.

210 peptides was measured ,~.~ three stimulator cell concentrations: 4 × l0 s, 2 x l0 s and 1 × l0 s cells per micr'o-titer well. farget concentration was constant (1 x 104 cells per well). The mean lysis obtained with each stimulator cell c,o ncentration for MV and a particular polypeptide is shown in Fig. 2. This response was consistently lower in the MS patients than that in the controls and it decreased with decreasing stimulator cell concentration. The CTL response for MV and N C was significendy different ( P < 0.05) between MS patients and controls at a responder cell concentration of 2 x 10 s cells/well (Fig. 2).

Diseussioa Recently, a substantial number of MS patients have been shown to have a reduced ability to generate CTLs specific for MV. This to date is the only antigen-specific immunological abnormality reported in this disease (Jacobson et al., 1985). The mechanism underlying this abnormality in the cellular immune response to MV is not yet understood. Several possibilities exist including seques~ tration of MV-CTLs within the CNS secondary to recognition of MV or cross-reactivity with myelin antigens, suppression, lack of T-cell repertoire, or an abnormality in the generation or maintenance of this component of the ~,.eRularimmune response to MV. The latter possibility could reflect either a MN-specific abnorw~4ity or a more generalized abnormality in the generation of class II HLArestricted C I L s which represent the major component of MV-CTLs in humans (J~cobson et al., 1984). In healthy individuals, a cellular immune respouse can be generated to each of the major polypeptides of the MV as measured by lymphoproliferation and in vitro generation of cytotoxic T-cell responses in bulk cultures (Jacobson et al., 1987). Understanding the profile of reactivity with respect to the components of MV in MS could be helpful in separating some of the possible causes for the reduced MV-specific CTLs in MS. In this study it was demonstrated that the reduced MV-CTLs in MS are due to a reduction in the response to at least two components of the virus: HA and NC. The response to F was also

r ~ u c e d but to a lesser extent. These findings have several implications regarding the mechanisms for the reduction in MV-CTL in MS. A possible defect in the T-cell repertoire is not supported by the findings in this study: Both healthy controls and MS patients with a normal MV-CTL response can generate CTL to a ~. least three components of MV and those with a reduced CTL response have a reduction in the CTL response to at least two components. Thus, a defect in the T-cell repertoire to three distinct MV antigens is unlikely. In addition, MS patients with low MV-CTL can generate an LPR to MV a~td have elevated levels of antibodies to MV and its polypeptides (unpublished data) which indicate adequate recognition of MV antigens by the T-cell antigen receptor. Because of the results of the limiting dilution experiment in this study, suppression seems an unlikely cause for the reduced MV-CTL in MS. Decreasing effector to target cells concentration would be expected to diminish suppressor cell activity and result in a parallel irl¢~ease in the MV-CTL response. In MS patients, MV-CTL response decreased With decreasing concentration of effector cells stimulated with either MV, HA, F or NC. This makes the possibility of a suppressor cell response to either MV or one of its polypeptide components unlikely. Sequestration of MV CTLs within the CNS could result in reduction of those CTLs in the peripheral blood. Sequestration would be consistent with the histopathological findings of inflammatory cell infdtrates in active MS lesions (Goodman and McFarlin, 1987). The majority of MV-CTLs are CD4 +. Sequestration would be likely if the majority of the T-cell infiltrates in tl:~: MS lesion were primarily CD4+; however, there is not an agreement about this (Zweiman and Lisak, 1980; Booss et ai., 1983; Traugott e" al., 1983; Hanser et al., 1986). Sequestration of MV-CTLs within the CNS could result from recognition of MV antigens within the CNS or alternatively from recognition of CNS antigens that cross-react with MV. While, attempts to detect viral antigens in MS brain have been unsuccessful, the studies by Rastogi et al. (1979) and Friedman et al. (1987) suggest cross-reactivity between MV and brain tissue from MS patients using serological assays.

If sequestration were to be due to recognition of MV antigens within the CNS, a reduction in CTL to several components of MV would be expected, whereas sequestration due to cross-reacting antigens would likely involve reduction to a single component of the virus. The observation in this study that there is a reduced CTL to more than one component of MV would indicate that if MV-CI'Ls were to be sequestered within the CNS, this is more likely to be due to recognition of MV antigem, rather than cross-reactivity among MV and CNS antigens. Moreover, experimental evidence in animals does not support cellular crossreactivity between MV and myelin basic protein (Liebert et al., 1988) and minimal sequence homology was found between MV polypeptides and myelin (Jahnke et al., 1985). Analysis of spinal fluid and CNS inflammatory cells fol CTL specific to MV a~d other viruses may contribute to tinderstanding the mechanisms involved in the reduction of MV-CTLs in MS. A fourth mechanism could involve reduction in a subset of CIM + cells that mediate class ll-restricted CTL function. This is supported by reports suggesting abnorma~i!ies in the CD4 ÷ T-cell subset in MS (Chofflon et al., 1988; Rose et al., 1988). MV-CTL is class II restricted (Jacobson et al., 1984). Evaluation of CTL function for other viruses that are class II-restricted (e.g., herpes virus) should help determine whether the defect in MS is MV-specific or is a reflection of a generalized defect in class ll-restricted CTL function. A deficiency in cytotoxic T-cells has been described in autoimmune diabetes in rats and the mechanism appears to be related to lack of maturation or destruction of CTLs rather than organ sequestration (Woda et al., 1986). In MS, the reduced MV-CTL response could be related to autoimmune mechanisms resulting in destruction of those CTLs. Finally, the reduced MV-CTL response in MS could be due to abnormalities in the induction or maintenance of this response. This could he related to infection with a defective strain of MV, severity and age at which the infection occurs. Although the mechanisms responsible for longterm immunity to MV are not understood, immunity is probably maintained through virus persistence. It is possible that in MS patients MV is

cleared or altered in such a way ff~at it affects the generation of the CTL componem of cell-mediated immunity. A reduced MV-CTL response has been found in three of four patients with sub.'umm ~lerosing panencephalitis (SSPE), a disease caused by a persistent MV infection (Dhib-Jalbut et al., 1987). Although the pathogenetic mechanisms in MS and SSPE are different, it can be speculated that the reduced MV-CTL response in both conditions is related to a persistent infection with MV. The manifestation of this persistent infection could be influenced by other factors. One possible factor is the presence of MV antibodies at the age at which the MV infection occurred. In case-controlled studies, patients with MS were found to have measles infection at a l a t e age (Sullivan et al., 1984) where MV antibodies could be present due to prior subclinical infection. In SSPE, the majority of patients bad measles infection under 2 years of age (Dhib-Jalbut et al., 1983) when maternal MV antibodies could still be present in the serum of those patients. Thus, the age at which measles infection is acquired or the presence of MV antibodies at the time of MV infection could influence long-term immunity to the virus. A

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