- Email: [email protected]
T cell vaccination in monoclonal antibody-induced hyperacute experimental allergic encephalomyelitis
Journal of Neuroimmunology, 24 (1989) 233-238
233
Elsevier JNI 00836
T cell vaccination in monoclonal antibody-induced hyperacute experimental allergic encephalomyelitis H e r m a n n J. S c h l u e s e n e r a n d R i c h a r d M e y e r m a n n Clinical Research Unit for Multiple Sclerosis, Max-Planck-Society, D-8700 Wiirzburg, F.R.G.
(Received 17 November 1988) (Revised, received18 April 1989) (Accepted 18 April 1989)
Key words: T cell vaccination; Demyelination; Myelin/oligodendrocyteglycoprotein
Summary Demyelinating inflammatory disease of the central nervous system (CNS) can be a multifactorial process mediated by cellular and antibody-mediated immune processes. In rats, hyperacute disease progression and severe demyelination can be induced in experimental allergic encephalomyelitis (EAE)diseased animals by injection of a monoclonal antibody, 8-18C5, specific for an oligodendrocyte cell surface glycoprotein. Here we demonstrate that this antibody-induced hyperacute EAE can be prevented by 'vaccination' with myelin basic protein (MBP)-specific T cells. Thus, the 8-18C5 antibody-mediated disease process is critically dependent on inflammatory processes induced by T lymphocytes and T cell vaccination is highly effective in preventing the development of demyelinating CNS lesions.
Introduction
In experimental allergic encephalomyelitis (EAE) antibodies against central nervous system (CNS) antigens considerably modulate the disease process. It has been described recently (Schluesener et al., 1987; Linington et al., 1988) that a mouse monoclonal IgG 1 antibody, 8-18C5, specific for myelin/oligodendrocyte glycoprotein (MOG; Linington et al., 1984) induces hyperacute disease progression and severe CNS demyelination. The
Address for correspondence: H.J. Schluesener,Clinical Research Unit for Multiple Sclerosis, Max-Planck-Society,JosefSchneider-Str. 11, D-8700 Wiirzburg, F.R.G.
essential contribution of both encephalitogenic T cells and 8-18C5 antibody to establish demyelinating lesions in the rat CNS has been clearly established (Schluesener et al., 1987, 1988; Lassmann et al., 1988; Linington et al., 1988). A promising therapeutic approach in the treatment of T ceilmediated autoimmune disease is vaccination with attenuated autoantigen-specific T cells. This experimental therapy has proven to be successful in autoimmune thyroiditis (EAT) (Maron et al., 1985), adjuvant arthritis (AA) (Lider et al., 1984, 1988) and EAE (Ben-Nun et al., 1981; Ben-Nun and Cohen, 1982; Holoshitz et al., 1983; Schluesener and Wekerle, 1985). We therefore evaluated the efficacy of T cell vaccination in 8-18C5 antibody-enhanced hyperacute rat demyelinating EAE.
0165-5728/89/$03.50 © 1989 ElsevierScience Publishers B.V. (Biomedical Division)
234
were harvested and incorporated radioactivity dctermined by liquid scintillation counting.
Material and methods
Induction of autoimmune disease Lewis rats (180-220 g body weight) were obtained from Charles River Laboratories and Zentralinstitut fiir Versuchtierkunde, Hannover, F.R.G. The procedures for isolation and maintenance of T lymphocyte lines S179/2(MBP) specific for MBP and S188 (Ova) specific for ovalbumin have been described (Schluesener et al., 1986). Line cells were expanded in interleukin-2 (IL-2) containing propagating Eagle's medium (Ben-Nun et al., 1981). Every 1-2 weeks cells were restimulated by incubation with antigen and irradiated syngeneic accessory cells for 3 days. Cells were then either used for experiments or propagated further in cell culture. For vaccination of rats, S179/2(MBP) and S188(Ova) line cells were restimulated as described above, fixed in 0.25% glutaraldehyde, washed twice in phosphatebuffered saline (PBS) and i.p. injected into Lewis rats in doses of 5 × 106 cells/rat (in 1 ml PBS). Rats were immunized with myelin basic protein/ complete Freund's adjuvant ( M B P / C F A ) (100/~1 of an emulsion of equal volumes of 10 m g / m l guinea pig MBP in PBS and Freund's adjuvant containing 10 m g / m l Mycobacteriurn tuberculosis H37 RA (Difco Laboratories, Detroit, MI, U.S.A.).
Purification and injection of antibody Hybridoma cell line 8-18C5 was grown in RPMI 1640 supplemented with 10% fetal calf serum. The antibody was purified from cell culture supernatants by immunoaffinity chromatography on columns of rabbit anti-mouse-lg (Tago, Burlingame, CA, U.S.A.). Prior to coupling these antisera to Tresyl-activated Sepharose 4B (Pharmacia, Piscataway, NJ, U.S.A.), antibodies cross-reacting with fetal calf serum were removed by solid-phase adsorption on columns of fetal calf serum/Sepharose 4B. Alternatively, 8-18C5 antibody was prepared from mouse serum and ascites by antiIgCl-agarose columns (Sigma, St. t,ouis, MO, U.S.A.). Antibody was buffered and concentrated on YM-5 membranes (Amicon Corp., Danvers, MA, U.S.A.). Antibody 8-18C5 was i.p. injected in a total volume of 1 ml PBS at a dose of 1 rag/rat.
Scoring of EA E EAE was scored as follows: (0) no disease: (1) decreased activity, limp tail: (2) mild paraparesis, unsteady gait; (3) moderate paraparesis, limbs are splayed apart, but voluntary movements still possible; (4) tetraplegia; (5) moribund.
Specificity of T cell line S179/2(MBP) Cells from line S179/2(MBP) were cocultured with antigen and antigen-presenting cells in round-bottomed microtiter plates (1 × ] 0 4 line cells/well, 1 × 106 2500 rad irradiated syngeneic thymocytes/well in 200 ~! RPMI 1640 with 1% rat serum and 10 g g / m l MBP). 48 h later, cells
Histological analysis Brain and spinal cord tissue samples were fixed in 4% paraformaldehyde, embedded in paraffin, and 8 #m sections were stained with Luxol fast blue or hematoxylin and eosin (HE) or a combination of both (LHE).
TABLE 1 A N T I G E N SPECIFICITY O F T CELL LINES T line cells (1 x 104) were stimulated with antigen (10 # g / m l MBP, 20 # g / m l ovalbumin) and 1 x 106 2500 rad X-irradiated, syngeneic thymocytes (in r o u n d - b o n o m e d microtiter plates in 200 #1 RPMI 1640 supplemented with 1% rat serum). 48 h later, cells were pulsed with 0.2 #Ci [3H]methyl-thymidine. After 18 h cells were harvested and incorporated radioactivity determined by liquid scintillation counting. Cultures were done in quadruplicates and data are given as means with standard deviations. Line
S179/2(MBP)
S188(Ova)
[ 3 HI Methyl-thymidine incorporation (cpm) MBP
Ovalbumin
Concanavalin A
No antigen
17 583 ± 1 284 316 ± 146
289 + 30 19201 :t: 1937
22 344 + 951 20016 __. 13012
248 + 31
512 ± 49
235 o
Results
c
4
F o r studying T cell v a c c i n a t i o n in hyperacute d e m y e l i n a t i n g EAE, two rat T cell lines were used. T h e first, S 1 7 9 / 2 ( M B P ) specific for MBP, the second, S188(Ova), specific for o v a l b u m i n (Table 1) are typical rat C D 4 ÷ T cells with the p h e n o t y p e W 3 / 1 3 *, W 3 / 2 5 +, O X - 8 - . Rats were vaccinated with 5 )< 10 6 f r e s h l y a c t i v a t e d a n d t h e n glutaraldehyde-fixed line cells. This fixation of line cells prevents the d e v e l o p m e n t of E A E after transfer of highly encephalitogenic S 1 7 9 / 2 ( M B P ) cells a n d makes it possible to store cells for prolonged periods of time. Rats were then i m m u n i z e d with M B P / C F A . H y p e r a c u t e disease progression was i n d u c e d by i.p. injection of 1 mg 8-18C5 a n t i b o d y on day 7 after i m m u n i z a t i o n with M B P / C F A . A detailed analysis of this model has been published elsewhere (Schluesener et al., 1987) a n d it should be noted that control a n t i b o d y injections were without a n y effects o n E A E (data n o t shown) (Schluesener et al., 1987). All rats from control groups - - not vaccinated with T line cells - - developed E A E (Table 2, Fig. 1). Injection of 8-18C5 a n t i b o d y i n d u c e d hyperacute E A E with first clinical signs already a p p e a r i n g o n day 10 a n d with all rats expiring by d a y 12 (Fig. l a ) . In c o m p a r i s o n , rats i m m u n i z e d with M B P / C F A developed a milder disease, p r e s e n t i n g with n e u r o logic signs on day 11 a n d recovering from disease by day 16 (Fig. l b ) . Vaccination with glutaraldehyde-fixed MBPspecific T cells from line S 1 7 9 / 2 ( M B P ) effectively prevented d e v e l o p m e n t of hyperacute as well as
•
o•
e•
s
Jr • • • • • ° • • • • • d
ooo
it • •
P [
eo
!
o
o
~ooo
[ ooo
.............
~ ocooooooooo
~' '
o
~ o o o
Fig. 1. Rats were randomly asigned to groups and pretreated as follows: rats from groups a and b received a control injection (1 ml PBS), groups c and d received 5 X I 0 6 glutaraldehydefixed cells from MBP-specific T cell line S179/2(MBP) and groups e and f received 5 X 1 0 6 glutaraldehyde-fixed cells from line S188(Ova). Two days later, all rats were immunized with MBP/CFA. To induce hyperacute disease progression, rats from groups a, c, and e (closed symbols) received i.p. injection of 1 mg 8-18C5 antibody (in 1 ml PBS). Control rats -- not immunized with MBP/CFA -- receiving only antibody 8-18C5 did show any signs of disease (Table 2 and Schluesener et al., 1987), nor did injection of unmunoglobulin with irrelevant specificity influence disease course (data not shown and Schluesener et al., 1986, 1987). c o n v e n t i o n a l EAE. V a c c i n a t i o n with o v a l b u m i n specific T cells from line $188(Ova) (Table 2, Fig. 1) did n o t p r e v e n t d e v e l o p m e n t of EAE. Histological o b s e r v a t i o n s c o n f i r m e d that the p r e v e n t i o n of clinical signs of E A E is associated with a suppression of i n f l a m m a t o r y cell infiltration into the CNS. In E A E rats treated with 8-18C5 a n t i b o d y large areas of i n f l a m m a t o r y lesion were f o u n d in b r a i n a n d spinal cord (Figs. 2 a n d 3). In contrast, rats vaccinated with S 1 7 9 / 2 ( M B P ) cells had
Rats were vaccinated with S179(MBP) and S188(Ova) line cells and immunized as described in in the legend to Fig. 1.
S179/2(MBP) S179/2(MBP) S188(Ova) $188(Ova)
8-18C5 MBP/CFA + 8-18C5 MBP/CFA MBP/CFA + 8-18C5 MBP/CFA MBP/CFA + 8-18C5 MBP/CFA
i
"
DOys FOLlOWm9 Immunlzollon ~/I~ M B P oad CFA
T CELL VACCINATION IN HYPERACUTE DEMYELINATING RAT EAE
EAE induction
ooo
' ; ' ' ' " ' ' ¢o. . . . ,~'
TABLE 2
Vaccination
o o
Disease/ total
Day of onset
score
0/5 8/8 8/8 0/11 0/11 8/8 8/8
10 11 10 11
0 5+0 2.8 + 0.5 0 0 5+0 3±0
Mean maximum
236
Fig. 2. The pathological changes of hyperacute EAE induced by injection of 8-18C5 antibody in EAE rats are (a) characterized by infiltration of numerous polymorphonuclear cells. Tissue inflammation is not limited to the perivascular area. Inflammatory cells diffusely infiltrate the parenchyma of both the gray and white matter, a and b: Lumbar spinal cord of non-vaccinated EAE rats (HE staining; bar in a = 30 ,am; bar in b = 100 gm).
neither inflammatory nor demyelinating foci in the C N S (Fig. 3). N o persistent subclinical pathological changes were found in the lumbar and sacral spinal cord or the medulla - - regions which have normally the highest incidence of lesions.
Discussion
E A E is considered to be an animal model for the h u m a n C N S demyelinating disease multiple sclerosis (MS) (Lassman, 1983; Alvord et al., 1984). The contribution of antibodies to demyelinating disease has long been a matter of debate (Brosnan
et al., 1977; Seil, 1977; Lassmann et al., 1981; R~Sytta et al., 1985; Tabira and Endoh, 1985; Trotter et al., 1986) and only recently monoclonal antibodies have been described inducing or amplifying i n f l a m m a t o r y demyelination in vivo (Fierz et al., 1987; Schluesener et al., 1987; Linington et al., 1988). The newly described model of 8-18C5 a n t i b o d y - i n d u c e d h y p e r a c u t e demyelinating rat E A E offers the o p p o r t u n i t y to study the interdependence of antibody-induced and cell-mediated processes. The observation that rats recovering from E A E are resistant to a further induction of disease by i m m u n i z a t i o n with M B P / C F A has been reported by several investi-
237
D
~
"~
~
., " : " " ~.~,~
~
.
~:" ";' :3.-" "
,
~
". 1~, ~ , ~ l : I r ' . : r ~
~
i •
~
Fig. 3. EAE rats were treated with 8-18C5 antibody as described in the legend to Fig. 1. The rats were killed 11 days after immunization for histology. EAE rats vaccinated with S179/2(MBP) T cells neither developed clinical signs (Fig. lc) nor pathological changes (Fig. 3a). In contrast, 8-18C5 antibody-reduced hyperacute EAE lesions in rats not vaccinated were scattered all over the spinal cord (Fig. 3b). Both pictures are from the lumbar region of the spinal cord (HE staining; bars = 300 #m). gators (Ben-Nun et al., 1981, 1982; Driscoll et al., 1982; Schluesener and Wekerle, 1985). Even injection of subclinical doses of encephalitogenic T cells has some protective effect. Disease induction by the vaccinating T cells can be circumvented by glutaraldehyde fixation of cells. However, such fixed cells are still capable to induce protective immunity. Several mechanisms have been proposed to explain this induction of resistance to a u t o i m m u n e disease (Swirkosz and Swanborg, 1977; Welch et al., 1977; Ellermann et al., 1988). Notably, the generation of C D 8 ÷ cytotoxic T cells with specificity for the disease-inducing C D 4 ÷ cell population has been demonstrated in rat E A E (Lider et al., 1988; Sun et al., 1988).
In comparison, T cell vaccination appears to be more efficient than monoclonal antibody-mediated therapy in hyperacute E A E (Schluesener et al., 1988). A l t h o u g h this monoclonal a n t i b o d y reduced lethality of hyperacute demyelinating E A E (Schluesener et al., 1988), even a few T cells escaping the cytotoxic effect of antibody treatment might be sufficient in inducing changes in b l o o d - b r a i n barrier permeability and in creating the i n f l a m m a t o r y milieu for development of demyelinating lesions. Activation of immune-regulatory processes by specific vaccination with autoi m m u n e T cells might generate physiologic suppressor mechanisms that appear to be more effective in preventing the activation and elimination of a u t o i m m u n e C D 4 ÷ T lymphocytes. In conclusion, T cell vaccination is a method with great promise in the treatment of a u t o i m m u n e disease. In addition, the p h e n o m e n o n itself will lead way to analysis of cellular networks and their pathological regulation during autoimmunity. Molecular biological and cellular immunological methods will greatly contribute to produce recombinant autoantigens, thus making it possible to produce T cells for even very rare antigens and to construct highly efficient vaccines against a u t o i m m u n e disease.
References Alvord, E.C., Kies, M.W. and Suckling, A.E. (1984) EAE -- A Useful Model for Multiple Sclerosis. Alan Liss, New York. Ben-Nun, A. and Cohen, I.R. (1982) Spontaneous remission and acquired resistance to autoimmune encephalomyelitis (EAE) are associated with suppression of T cell reactivity: suppressed EAE effector cells recovered as T cell lines. J. Immunol. 128, 1450-1457. Ben-Nun, A., Wekerle, H. and Cohen, I.R. (1981) The rapid isolation of clonable antigen-specific T lymphocyte lines capable of mediating autoimmune encephalomyelitis. Eur. J. lmmunol. 11, 195-199. Brosnan, C.F., Stoner, G.L., Bloom, B.L. and Wisniewski, H.M. (1977) Studies on demyelination by activated lymphocytes in the rabbit eye. II. Antibody-dependent ceU-mediated demyelination. J. Immunol. 11, 2109-2110. Driscoll, B.F., Kies, M.W. and Alvord, E.C. (1982) Suppression of autoimmune experimental allergic encephalomyelitis in guinea pigs by prior transfer of suboptimal numbers of EAE-effector cells: induction of chronic EAE in whole tissue-sensitized guinea pigs. J. Immunol. 128, 635-638.
238 Ellermann, K.E, Powers, J.M. and Brostoff, S.W. (1988) A suppressor T-lymphocyte cell line for autoimmune encephalomyelitis. Nature 331,265-267. Fier'z, W., Heininger, K., Schaefer, B., Toyka, K.V., Linington, C. and Lassmann, H. (1987) Synergism in the pathogenesis of EAE induced by an MBP-specific T cell line and monoclonal antibodies to galactocerebroside or to a myelin/ oligodendrocyte glycoprotein. J. Neuroimmunol. 16, 55-56. Holoshitz, J.. Frnekel, A., Ben-Nun, A. and Cohen, I. (1983) Autoimmune enecephalomyelitis (EAE) mediated or prevented by T lymphocyte lines directed against diverse antigenic determinants of myelin basic protein. Vaccination is determinant specific. J. Immunol. 131, 2810-1813. Lassmann, H. (1983) Chronic relapsing experimental allergic encephalomyelitis: its value as an experimental model for multiple sclerosis. J. Neurol. 229, 207-220. Lassmann, H., Kitz, K. and Wisniewski, H.M. (1981) In vivo effect of sera from animals with chronic relapsing experimental allergic encephalomyelitis on central and peripheral myelin. Acta Neuropathol. 55, 297-302. Lassmann, H., Brunner, C., Bradl, M. and Linington, C. (1988) EAE: the balance between encephalitogenic T lymphocytes and demyelinating antibodies determines size and structure of demyelinating lesions. Acta Neuropathol. 75, 566-576. Lider, O., Karin, N., Shinitzky, J. and Cohen, I.R. (1984) Therapeutic vaccination against adjuvant arthritis using autoimmune T cells treated with hydrostatic pressure. Proc. Natl. Acad. Sci. U.S.A. 84, 4577-4580. Lider, O., Reshef, T., Beraud, E., Ben-Nun, A. and Cohen, I. (1988) Anti-idiotypic network induced by T cell vaccination against experimental autoimmune encephalomyelitis. Science 239, 181-183. l,inington, C., Webb, M. and Woodhams, P.L. (1984) A novel myelin-associated glycoprotein defined by a mouse monoclonal antibody. J. Neuroimmunol. 6, 387-396. Linington, C., Bradl, M., Lassmann, H., Brunner. C. and Vass, K. (1988) Augmentation of demyelination in rat acute allergic encephalomyelitis by circulating mouse monoclonal antibodies against a myelin/oligodendrocyte glycoprotein. Am. J. Pathol. 130, 443-454. Maron. R., Zerubavel, R., Friedman, A. and Cohen, I.R. (1985) T lymphocyte line specific for thyroglobulin produces or vaccinates against autoimmune thyroiditis in mice. J. Immunol. 131, 2316-2322. Rt~ytta, M., Lyman, W.D., Roth, G.A., Bomstein, M.B. and Raine, C.S. (1985) Preliminary analysis of cell and seruminduced demyelination in vitro using a syngeneic system. Acta Neurol. Scand. 71,226-325.
Schluesener, H.J. (1986) Inhibition of rat autoimmune 1- cell activation by monoclonal antibodies. J. Neuroimmunol. 11, 261 - 270. Schluesener. H.J. and Wekerle, H. (1985) Autoaggressive T lymphocyte lines recognizing the encephalitogenic region of myelin basic protein: in vitro selection from unprimed rat T lymphocyte populations. J. lmmunol. 135, 3128-3133. Schluesener, H.J., Brunner, C., Vass, K. and Lassmann, H. (1986) Therapy of rat autoimmune disease by a monoclonal antibody specific for T lymphoblasts. J. Immunol. 137, 3814-3820. Schluesener, H.J., Sobel, R., Linington, C. and Weiner. H. (1987) A monoclonal antibody against a myelin/oligodendrocyte glycoprotein induces relapses and demyelination in central nervous system autoimmune disease. J. Immunol. 139, 2505-2511. Schluesener, H.J., Sobel, R.A. and Weiner, H.L. (1988) I)emyelinating experimental allergic encephalomyelitis (EAEI in the rat: treatment with a monoclonal antibody against activated T cells. J. Neuroimmunol. 18, 341-351. Schluesener, H.J., Lider, O. and Sobel, R.A. (1989) Induction of hyperacute brain inflammation and demyelination by activated encephalitogenic T cells and a monoclonal antibody specific for a myelin/oligodendrocyte glycoprotein. Autoimmunity 2, 265-273. Seil, F.J. (1977) Tissue culture studies of demylinating disease: a critical review. Ann. Neurol. 2, 345-357. Sun, D., Qin, Y., Chluba, J., Epplen, J.T. and Wekerle. It. (1988) Suppression of experimentally induced autoimmune encephalomyelitis by cytolitic T - T cell interactions. Nature 332, 843-845. Swirkosz, J.E. and Swanborg, R.H. (1977) lmmunoregulation of experimental allergic encephalomyelitis: conditions for induction of suppressor cells and analysis of mechanisms. J. lmmunol. 119, 1501-1506. Tabira, T. and Endoh, M. (1985) Humoral immune response to myelin basic protein, cerebroside and ganglioside in chronic relapsing experimental allergic encephalomyelitis in the guinea pig. J. Neurol. Sci. 67, 201-209. Trotter, J., DeJong, L.J. and Smith, M.E. (1986) Opsonization with antimyelin antibody increases uptake and intracellular metabolism of myelin in inflammatory macrophages. J. Neurochem. 47, 779-785. Welch, A.M., Holda, J.H. and Swanborg, R.H. (1977) Regulation of experimental allergic encephalomyelitis. I1. Appearance of suppressor cells during the remission phase of the disease. J. Immunol. 125, 186-189.
233
Elsevier JNI 00836
T cell vaccination in monoclonal antibody-induced hyperacute experimental allergic encephalomyelitis H e r m a n n J. S c h l u e s e n e r a n d R i c h a r d M e y e r m a n n Clinical Research Unit for Multiple Sclerosis, Max-Planck-Society, D-8700 Wiirzburg, F.R.G.
(Received 17 November 1988) (Revised, received18 April 1989) (Accepted 18 April 1989)
Key words: T cell vaccination; Demyelination; Myelin/oligodendrocyteglycoprotein
Summary Demyelinating inflammatory disease of the central nervous system (CNS) can be a multifactorial process mediated by cellular and antibody-mediated immune processes. In rats, hyperacute disease progression and severe demyelination can be induced in experimental allergic encephalomyelitis (EAE)diseased animals by injection of a monoclonal antibody, 8-18C5, specific for an oligodendrocyte cell surface glycoprotein. Here we demonstrate that this antibody-induced hyperacute EAE can be prevented by 'vaccination' with myelin basic protein (MBP)-specific T cells. Thus, the 8-18C5 antibody-mediated disease process is critically dependent on inflammatory processes induced by T lymphocytes and T cell vaccination is highly effective in preventing the development of demyelinating CNS lesions.
Introduction
In experimental allergic encephalomyelitis (EAE) antibodies against central nervous system (CNS) antigens considerably modulate the disease process. It has been described recently (Schluesener et al., 1987; Linington et al., 1988) that a mouse monoclonal IgG 1 antibody, 8-18C5, specific for myelin/oligodendrocyte glycoprotein (MOG; Linington et al., 1984) induces hyperacute disease progression and severe CNS demyelination. The
Address for correspondence: H.J. Schluesener,Clinical Research Unit for Multiple Sclerosis, Max-Planck-Society,JosefSchneider-Str. 11, D-8700 Wiirzburg, F.R.G.
essential contribution of both encephalitogenic T cells and 8-18C5 antibody to establish demyelinating lesions in the rat CNS has been clearly established (Schluesener et al., 1987, 1988; Lassmann et al., 1988; Linington et al., 1988). A promising therapeutic approach in the treatment of T ceilmediated autoimmune disease is vaccination with attenuated autoantigen-specific T cells. This experimental therapy has proven to be successful in autoimmune thyroiditis (EAT) (Maron et al., 1985), adjuvant arthritis (AA) (Lider et al., 1984, 1988) and EAE (Ben-Nun et al., 1981; Ben-Nun and Cohen, 1982; Holoshitz et al., 1983; Schluesener and Wekerle, 1985). We therefore evaluated the efficacy of T cell vaccination in 8-18C5 antibody-enhanced hyperacute rat demyelinating EAE.
0165-5728/89/$03.50 © 1989 ElsevierScience Publishers B.V. (Biomedical Division)
234
were harvested and incorporated radioactivity dctermined by liquid scintillation counting.
Material and methods
Induction of autoimmune disease Lewis rats (180-220 g body weight) were obtained from Charles River Laboratories and Zentralinstitut fiir Versuchtierkunde, Hannover, F.R.G. The procedures for isolation and maintenance of T lymphocyte lines S179/2(MBP) specific for MBP and S188 (Ova) specific for ovalbumin have been described (Schluesener et al., 1986). Line cells were expanded in interleukin-2 (IL-2) containing propagating Eagle's medium (Ben-Nun et al., 1981). Every 1-2 weeks cells were restimulated by incubation with antigen and irradiated syngeneic accessory cells for 3 days. Cells were then either used for experiments or propagated further in cell culture. For vaccination of rats, S179/2(MBP) and S188(Ova) line cells were restimulated as described above, fixed in 0.25% glutaraldehyde, washed twice in phosphatebuffered saline (PBS) and i.p. injected into Lewis rats in doses of 5 × 106 cells/rat (in 1 ml PBS). Rats were immunized with myelin basic protein/ complete Freund's adjuvant ( M B P / C F A ) (100/~1 of an emulsion of equal volumes of 10 m g / m l guinea pig MBP in PBS and Freund's adjuvant containing 10 m g / m l Mycobacteriurn tuberculosis H37 RA (Difco Laboratories, Detroit, MI, U.S.A.).
Purification and injection of antibody Hybridoma cell line 8-18C5 was grown in RPMI 1640 supplemented with 10% fetal calf serum. The antibody was purified from cell culture supernatants by immunoaffinity chromatography on columns of rabbit anti-mouse-lg (Tago, Burlingame, CA, U.S.A.). Prior to coupling these antisera to Tresyl-activated Sepharose 4B (Pharmacia, Piscataway, NJ, U.S.A.), antibodies cross-reacting with fetal calf serum were removed by solid-phase adsorption on columns of fetal calf serum/Sepharose 4B. Alternatively, 8-18C5 antibody was prepared from mouse serum and ascites by antiIgCl-agarose columns (Sigma, St. t,ouis, MO, U.S.A.). Antibody was buffered and concentrated on YM-5 membranes (Amicon Corp., Danvers, MA, U.S.A.). Antibody 8-18C5 was i.p. injected in a total volume of 1 ml PBS at a dose of 1 rag/rat.
Scoring of EA E EAE was scored as follows: (0) no disease: (1) decreased activity, limp tail: (2) mild paraparesis, unsteady gait; (3) moderate paraparesis, limbs are splayed apart, but voluntary movements still possible; (4) tetraplegia; (5) moribund.
Specificity of T cell line S179/2(MBP) Cells from line S179/2(MBP) were cocultured with antigen and antigen-presenting cells in round-bottomed microtiter plates (1 × ] 0 4 line cells/well, 1 × 106 2500 rad irradiated syngeneic thymocytes/well in 200 ~! RPMI 1640 with 1% rat serum and 10 g g / m l MBP). 48 h later, cells
Histological analysis Brain and spinal cord tissue samples were fixed in 4% paraformaldehyde, embedded in paraffin, and 8 #m sections were stained with Luxol fast blue or hematoxylin and eosin (HE) or a combination of both (LHE).
TABLE 1 A N T I G E N SPECIFICITY O F T CELL LINES T line cells (1 x 104) were stimulated with antigen (10 # g / m l MBP, 20 # g / m l ovalbumin) and 1 x 106 2500 rad X-irradiated, syngeneic thymocytes (in r o u n d - b o n o m e d microtiter plates in 200 #1 RPMI 1640 supplemented with 1% rat serum). 48 h later, cells were pulsed with 0.2 #Ci [3H]methyl-thymidine. After 18 h cells were harvested and incorporated radioactivity determined by liquid scintillation counting. Cultures were done in quadruplicates and data are given as means with standard deviations. Line
S179/2(MBP)
S188(Ova)
[ 3 HI Methyl-thymidine incorporation (cpm) MBP
Ovalbumin
Concanavalin A
No antigen
17 583 ± 1 284 316 ± 146
289 + 30 19201 :t: 1937
22 344 + 951 20016 __. 13012
248 + 31
512 ± 49
235 o
Results
c
4
F o r studying T cell v a c c i n a t i o n in hyperacute d e m y e l i n a t i n g EAE, two rat T cell lines were used. T h e first, S 1 7 9 / 2 ( M B P ) specific for MBP, the second, S188(Ova), specific for o v a l b u m i n (Table 1) are typical rat C D 4 ÷ T cells with the p h e n o t y p e W 3 / 1 3 *, W 3 / 2 5 +, O X - 8 - . Rats were vaccinated with 5 )< 10 6 f r e s h l y a c t i v a t e d a n d t h e n glutaraldehyde-fixed line cells. This fixation of line cells prevents the d e v e l o p m e n t of E A E after transfer of highly encephalitogenic S 1 7 9 / 2 ( M B P ) cells a n d makes it possible to store cells for prolonged periods of time. Rats were then i m m u n i z e d with M B P / C F A . H y p e r a c u t e disease progression was i n d u c e d by i.p. injection of 1 mg 8-18C5 a n t i b o d y on day 7 after i m m u n i z a t i o n with M B P / C F A . A detailed analysis of this model has been published elsewhere (Schluesener et al., 1987) a n d it should be noted that control a n t i b o d y injections were without a n y effects o n E A E (data n o t shown) (Schluesener et al., 1987). All rats from control groups - - not vaccinated with T line cells - - developed E A E (Table 2, Fig. 1). Injection of 8-18C5 a n t i b o d y i n d u c e d hyperacute E A E with first clinical signs already a p p e a r i n g o n day 10 a n d with all rats expiring by d a y 12 (Fig. l a ) . In c o m p a r i s o n , rats i m m u n i z e d with M B P / C F A developed a milder disease, p r e s e n t i n g with n e u r o logic signs on day 11 a n d recovering from disease by day 16 (Fig. l b ) . Vaccination with glutaraldehyde-fixed MBPspecific T cells from line S 1 7 9 / 2 ( M B P ) effectively prevented d e v e l o p m e n t of hyperacute as well as
•
o•
e•
s
Jr • • • • • ° • • • • • d
ooo
it • •
P [
eo
!
o
o
~ooo
[ ooo
.............
~ ocooooooooo
~' '
o
~ o o o
Fig. 1. Rats were randomly asigned to groups and pretreated as follows: rats from groups a and b received a control injection (1 ml PBS), groups c and d received 5 X I 0 6 glutaraldehydefixed cells from MBP-specific T cell line S179/2(MBP) and groups e and f received 5 X 1 0 6 glutaraldehyde-fixed cells from line S188(Ova). Two days later, all rats were immunized with MBP/CFA. To induce hyperacute disease progression, rats from groups a, c, and e (closed symbols) received i.p. injection of 1 mg 8-18C5 antibody (in 1 ml PBS). Control rats -- not immunized with MBP/CFA -- receiving only antibody 8-18C5 did show any signs of disease (Table 2 and Schluesener et al., 1987), nor did injection of unmunoglobulin with irrelevant specificity influence disease course (data not shown and Schluesener et al., 1986, 1987). c o n v e n t i o n a l EAE. V a c c i n a t i o n with o v a l b u m i n specific T cells from line $188(Ova) (Table 2, Fig. 1) did n o t p r e v e n t d e v e l o p m e n t of EAE. Histological o b s e r v a t i o n s c o n f i r m e d that the p r e v e n t i o n of clinical signs of E A E is associated with a suppression of i n f l a m m a t o r y cell infiltration into the CNS. In E A E rats treated with 8-18C5 a n t i b o d y large areas of i n f l a m m a t o r y lesion were f o u n d in b r a i n a n d spinal cord (Figs. 2 a n d 3). In contrast, rats vaccinated with S 1 7 9 / 2 ( M B P ) cells had
Rats were vaccinated with S179(MBP) and S188(Ova) line cells and immunized as described in in the legend to Fig. 1.
S179/2(MBP) S179/2(MBP) S188(Ova) $188(Ova)
8-18C5 MBP/CFA + 8-18C5 MBP/CFA MBP/CFA + 8-18C5 MBP/CFA MBP/CFA + 8-18C5 MBP/CFA
i
"
DOys FOLlOWm9 Immunlzollon ~/I~ M B P oad CFA
T CELL VACCINATION IN HYPERACUTE DEMYELINATING RAT EAE
EAE induction
ooo
' ; ' ' ' " ' ' ¢o. . . . ,~'
TABLE 2
Vaccination
o o
Disease/ total
Day of onset
score
0/5 8/8 8/8 0/11 0/11 8/8 8/8
10 11 10 11
0 5+0 2.8 + 0.5 0 0 5+0 3±0
Mean maximum
236
Fig. 2. The pathological changes of hyperacute EAE induced by injection of 8-18C5 antibody in EAE rats are (a) characterized by infiltration of numerous polymorphonuclear cells. Tissue inflammation is not limited to the perivascular area. Inflammatory cells diffusely infiltrate the parenchyma of both the gray and white matter, a and b: Lumbar spinal cord of non-vaccinated EAE rats (HE staining; bar in a = 30 ,am; bar in b = 100 gm).
neither inflammatory nor demyelinating foci in the C N S (Fig. 3). N o persistent subclinical pathological changes were found in the lumbar and sacral spinal cord or the medulla - - regions which have normally the highest incidence of lesions.
Discussion
E A E is considered to be an animal model for the h u m a n C N S demyelinating disease multiple sclerosis (MS) (Lassman, 1983; Alvord et al., 1984). The contribution of antibodies to demyelinating disease has long been a matter of debate (Brosnan
et al., 1977; Seil, 1977; Lassmann et al., 1981; R~Sytta et al., 1985; Tabira and Endoh, 1985; Trotter et al., 1986) and only recently monoclonal antibodies have been described inducing or amplifying i n f l a m m a t o r y demyelination in vivo (Fierz et al., 1987; Schluesener et al., 1987; Linington et al., 1988). The newly described model of 8-18C5 a n t i b o d y - i n d u c e d h y p e r a c u t e demyelinating rat E A E offers the o p p o r t u n i t y to study the interdependence of antibody-induced and cell-mediated processes. The observation that rats recovering from E A E are resistant to a further induction of disease by i m m u n i z a t i o n with M B P / C F A has been reported by several investi-
237
D
~
"~
~
., " : " " ~.~,~
~
.
~:" ";' :3.-" "
,
~
". 1~, ~ , ~ l : I r ' . : r ~
~
i •
~
Fig. 3. EAE rats were treated with 8-18C5 antibody as described in the legend to Fig. 1. The rats were killed 11 days after immunization for histology. EAE rats vaccinated with S179/2(MBP) T cells neither developed clinical signs (Fig. lc) nor pathological changes (Fig. 3a). In contrast, 8-18C5 antibody-reduced hyperacute EAE lesions in rats not vaccinated were scattered all over the spinal cord (Fig. 3b). Both pictures are from the lumbar region of the spinal cord (HE staining; bars = 300 #m). gators (Ben-Nun et al., 1981, 1982; Driscoll et al., 1982; Schluesener and Wekerle, 1985). Even injection of subclinical doses of encephalitogenic T cells has some protective effect. Disease induction by the vaccinating T cells can be circumvented by glutaraldehyde fixation of cells. However, such fixed cells are still capable to induce protective immunity. Several mechanisms have been proposed to explain this induction of resistance to a u t o i m m u n e disease (Swirkosz and Swanborg, 1977; Welch et al., 1977; Ellermann et al., 1988). Notably, the generation of C D 8 ÷ cytotoxic T cells with specificity for the disease-inducing C D 4 ÷ cell population has been demonstrated in rat E A E (Lider et al., 1988; Sun et al., 1988).
In comparison, T cell vaccination appears to be more efficient than monoclonal antibody-mediated therapy in hyperacute E A E (Schluesener et al., 1988). A l t h o u g h this monoclonal a n t i b o d y reduced lethality of hyperacute demyelinating E A E (Schluesener et al., 1988), even a few T cells escaping the cytotoxic effect of antibody treatment might be sufficient in inducing changes in b l o o d - b r a i n barrier permeability and in creating the i n f l a m m a t o r y milieu for development of demyelinating lesions. Activation of immune-regulatory processes by specific vaccination with autoi m m u n e T cells might generate physiologic suppressor mechanisms that appear to be more effective in preventing the activation and elimination of a u t o i m m u n e C D 4 ÷ T lymphocytes. In conclusion, T cell vaccination is a method with great promise in the treatment of a u t o i m m u n e disease. In addition, the p h e n o m e n o n itself will lead way to analysis of cellular networks and their pathological regulation during autoimmunity. Molecular biological and cellular immunological methods will greatly contribute to produce recombinant autoantigens, thus making it possible to produce T cells for even very rare antigens and to construct highly efficient vaccines against a u t o i m m u n e disease.
References Alvord, E.C., Kies, M.W. and Suckling, A.E. (1984) EAE -- A Useful Model for Multiple Sclerosis. Alan Liss, New York. Ben-Nun, A. and Cohen, I.R. (1982) Spontaneous remission and acquired resistance to autoimmune encephalomyelitis (EAE) are associated with suppression of T cell reactivity: suppressed EAE effector cells recovered as T cell lines. J. Immunol. 128, 1450-1457. Ben-Nun, A., Wekerle, H. and Cohen, I.R. (1981) The rapid isolation of clonable antigen-specific T lymphocyte lines capable of mediating autoimmune encephalomyelitis. Eur. J. lmmunol. 11, 195-199. Brosnan, C.F., Stoner, G.L., Bloom, B.L. and Wisniewski, H.M. (1977) Studies on demyelination by activated lymphocytes in the rabbit eye. II. Antibody-dependent ceU-mediated demyelination. J. Immunol. 11, 2109-2110. Driscoll, B.F., Kies, M.W. and Alvord, E.C. (1982) Suppression of autoimmune experimental allergic encephalomyelitis in guinea pigs by prior transfer of suboptimal numbers of EAE-effector cells: induction of chronic EAE in whole tissue-sensitized guinea pigs. J. Immunol. 128, 635-638.
238 Ellermann, K.E, Powers, J.M. and Brostoff, S.W. (1988) A suppressor T-lymphocyte cell line for autoimmune encephalomyelitis. Nature 331,265-267. Fier'z, W., Heininger, K., Schaefer, B., Toyka, K.V., Linington, C. and Lassmann, H. (1987) Synergism in the pathogenesis of EAE induced by an MBP-specific T cell line and monoclonal antibodies to galactocerebroside or to a myelin/ oligodendrocyte glycoprotein. J. Neuroimmunol. 16, 55-56. Holoshitz, J.. Frnekel, A., Ben-Nun, A. and Cohen, I. (1983) Autoimmune enecephalomyelitis (EAE) mediated or prevented by T lymphocyte lines directed against diverse antigenic determinants of myelin basic protein. Vaccination is determinant specific. J. Immunol. 131, 2810-1813. Lassmann, H. (1983) Chronic relapsing experimental allergic encephalomyelitis: its value as an experimental model for multiple sclerosis. J. Neurol. 229, 207-220. Lassmann, H., Kitz, K. and Wisniewski, H.M. (1981) In vivo effect of sera from animals with chronic relapsing experimental allergic encephalomyelitis on central and peripheral myelin. Acta Neuropathol. 55, 297-302. Lassmann, H., Brunner, C., Bradl, M. and Linington, C. (1988) EAE: the balance between encephalitogenic T lymphocytes and demyelinating antibodies determines size and structure of demyelinating lesions. Acta Neuropathol. 75, 566-576. Lider, O., Karin, N., Shinitzky, J. and Cohen, I.R. (1984) Therapeutic vaccination against adjuvant arthritis using autoimmune T cells treated with hydrostatic pressure. Proc. Natl. Acad. Sci. U.S.A. 84, 4577-4580. Lider, O., Reshef, T., Beraud, E., Ben-Nun, A. and Cohen, I. (1988) Anti-idiotypic network induced by T cell vaccination against experimental autoimmune encephalomyelitis. Science 239, 181-183. l,inington, C., Webb, M. and Woodhams, P.L. (1984) A novel myelin-associated glycoprotein defined by a mouse monoclonal antibody. J. Neuroimmunol. 6, 387-396. Linington, C., Bradl, M., Lassmann, H., Brunner. C. and Vass, K. (1988) Augmentation of demyelination in rat acute allergic encephalomyelitis by circulating mouse monoclonal antibodies against a myelin/oligodendrocyte glycoprotein. Am. J. Pathol. 130, 443-454. Maron. R., Zerubavel, R., Friedman, A. and Cohen, I.R. (1985) T lymphocyte line specific for thyroglobulin produces or vaccinates against autoimmune thyroiditis in mice. J. Immunol. 131, 2316-2322. Rt~ytta, M., Lyman, W.D., Roth, G.A., Bomstein, M.B. and Raine, C.S. (1985) Preliminary analysis of cell and seruminduced demyelination in vitro using a syngeneic system. Acta Neurol. Scand. 71,226-325.
Schluesener, H.J. (1986) Inhibition of rat autoimmune 1- cell activation by monoclonal antibodies. J. Neuroimmunol. 11, 261 - 270. Schluesener. H.J. and Wekerle, H. (1985) Autoaggressive T lymphocyte lines recognizing the encephalitogenic region of myelin basic protein: in vitro selection from unprimed rat T lymphocyte populations. J. lmmunol. 135, 3128-3133. Schluesener, H.J., Brunner, C., Vass, K. and Lassmann, H. (1986) Therapy of rat autoimmune disease by a monoclonal antibody specific for T lymphoblasts. J. Immunol. 137, 3814-3820. Schluesener, H.J., Sobel, R., Linington, C. and Weiner. H. (1987) A monoclonal antibody against a myelin/oligodendrocyte glycoprotein induces relapses and demyelination in central nervous system autoimmune disease. J. Immunol. 139, 2505-2511. Schluesener, H.J., Sobel, R.A. and Weiner, H.L. (1988) I)emyelinating experimental allergic encephalomyelitis (EAEI in the rat: treatment with a monoclonal antibody against activated T cells. J. Neuroimmunol. 18, 341-351. Schluesener, H.J., Lider, O. and Sobel, R.A. (1989) Induction of hyperacute brain inflammation and demyelination by activated encephalitogenic T cells and a monoclonal antibody specific for a myelin/oligodendrocyte glycoprotein. Autoimmunity 2, 265-273. Seil, F.J. (1977) Tissue culture studies of demylinating disease: a critical review. Ann. Neurol. 2, 345-357. Sun, D., Qin, Y., Chluba, J., Epplen, J.T. and Wekerle. It. (1988) Suppression of experimentally induced autoimmune encephalomyelitis by cytolitic T - T cell interactions. Nature 332, 843-845. Swirkosz, J.E. and Swanborg, R.H. (1977) lmmunoregulation of experimental allergic encephalomyelitis: conditions for induction of suppressor cells and analysis of mechanisms. J. lmmunol. 119, 1501-1506. Tabira, T. and Endoh, M. (1985) Humoral immune response to myelin basic protein, cerebroside and ganglioside in chronic relapsing experimental allergic encephalomyelitis in the guinea pig. J. Neurol. Sci. 67, 201-209. Trotter, J., DeJong, L.J. and Smith, M.E. (1986) Opsonization with antimyelin antibody increases uptake and intracellular metabolism of myelin in inflammatory macrophages. J. Neurochem. 47, 779-785. Welch, A.M., Holda, J.H. and Swanborg, R.H. (1977) Regulation of experimental allergic encephalomyelitis. I1. Appearance of suppressor cells during the remission phase of the disease. J. Immunol. 125, 186-189.