Ia restriction of murine encephalitogenic T-cell lines in vitro and in vivo

Journal of Neurotmmunology,

9 (1985) 281-291

281

Elsevier JNI 00294

Ia Restriction of Murine Encephalitogenic T-Cell Lines in Vitro and in Vivo K. Sakai, T. Namikawa, T. Kunishita, T. Yoshimura and T. Tabira National Center for Nervous, Mental and Muscular Disorders, 4 - 1 - 1 0 g a w a - Higasht - Machz, Kodaira, Tokyo 187 (Japan)

(Received 30 November, 1984) (Revised, received 13 February, 1985) (Accepted 13 February, 1985)

Summaff To clarify the functional role of the I region-associated (Ia) antigen in autoimmune central nervous system disorders, we generated long-term cultured lines of encephalitogenic T cells responsive to myelin basic protein from SJL strain mice (H-2 s) and investigated genetic restriction in proliferative and encephalitogenic activities of the lines. These cell lines bear a Lyt 1+,2 - phenotype, and show antigen-specific and I-As restricted proliferative responses in vitro. These lines induced full-blown experimental allergic encephalomyelitis (EAE) in immunocompromised recipients carrying the I-As genotype. These data demonstrate that encephalitogenic T lymphocytes recognize the antigen in combination with the Ia antigen to induce EAE. Key words:

Encephalomyelitis,

allergic -

Genetic restriction -

la antigen

-

T-cell

lines, e n c e p h a l i t o g e n i c

Introduction A large body of knowledge has been accumulated by experimental studies on the murine immune system. We now know that T cells recognize a specific antigen in

This work was partially supported by a grant from the Neuroimmunological Disorders Research Committee, the Ministry of Health and Welfare of Japan. Reprint requests: Dr. K. Sakai, National Center for Nervous, Mental and Muscular Disorders, 4-1-1 Ogawa-Higashi-Machi, Kodaira, Tokyo 187, Japan. 0165-5728/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

282 association with the major histocompatibility complex (MHC) and that products of MHC play functional roles in many aspects of the immune system. In experimental allergic encephalomyelitis (EAE), which is known to be mediated by T lymphocytes against myelin basic protein (MBP) (Driscoll and Kies 1979; Richert et al. 1979; Pettinelli and McFarlin 1981), it has been suggested that MHC products are involved in the pathogenetic mechanisms. An I region-associated (Ia) antigen appears in the central nervous system (CNS) at an early stage of EAE (Sobel et al. 1984) and a monoclonal antibody against a product of the I-A subregion suppresses induction of EAE (Steinman et al. 1981, 1983). We have recently generated T-cell lines against MBP derived from lymph node cells of SJL strain mice as previously reported in other animals (Ben-Nun et al. 1981; Ben-Nun and Cohen 1982; Ben-Nun and Lando 1983). The cell lines can adoptively transfer full-blown EAE to naive syngeneic mice with a relatively small number of cells, which made it easier '~o analyze the immune mechanisms of EAE. In this communication we show that the T-cell lines recognize MBP in the presence of antigen-presenting cells (APC) from strains of mice whose genotype of the I-A subregion is identical to that of the T-cell donor. The cell lines produce EAE only in recipients that have an Ia antigen identical to that of the donor mice.

Materials and Methods

Animals SJL mice were obtained from Gokita Bleeding Serivce, Tokyo, Japan. BALB/c, C3H/He and A / J mice were from Sankyo Laboratory Serivce, Tokyo, Japan. A.SW/Sn mice were purchased from Jackson Laboratories, Bar Harbor, ME, U.S.A. A.TL and DDD/1 mice were kindly supplied by Professor K. Yamanouchi (Institute of Medical Sciences, University of Tokyo). All mice were female and used at 6-8 weeks of age. A n tigens Myelin basic protein (MBP) was prepared from CNS tissues of the cow, the guinea pig and the rat according to the procedure described by Deibler et al. (1972). Purity of MBP was ascertained by SDS polyacrylamide gel electrophoresis. With rat MBP the small form was used throughout the experiments. Rabbit MBP was kindly provided by Dr. M.W. Kies (National Institutes of Health, Bethesda, MD, U.S.A.). Bovine proteolipid protein (PLP) was prepared from CNS tissues from the cow according to the procedure described by Folch et al. (1951) and purified by the method of Helynck et al. (1983). Preparation and culture of cells The long-term cultured T-cell lines were prepared as follows. SJL mice were immunized by subcutaneous injection of a total of 0.1 ml of an emulsion of 400 ~tg of rat MBP and 50/xg of Mycobacterium tuberculosis (H37Ra, Difco) in incomplete Freund's adjuvant distributed at four sites on the flank according to the protocol of

283 Brown and McFarlin (1981). Ten days later, single cell suspensions were made from the inguinal and axillary lymph nodes and cultured for 5 days with 100 /~g of rat MBP at a concentration of 4 x 106 cells/ml in a culture medium consisting of RPMI 1640, 1% fresh normal mouse serum, 100 units of penicillin/ml, 100 /zg of streptomycin/ml, 3 mM L-glutamine, and 5 X 10 -5 M 2-mercaptoethanol. Cells were collected after 5 days and cultured in the above medium but containing 5% fetal calf serum (FCS) instead of mouse serum and 15% concanavalin A (Con A) supernatant (Kruisbeek et al. 1980) and no antigen. Cells were maintained by alternate antigenic stimulation in the supplemented RPMI 1640 without Con A supernatant containing 1% fresh normal mouse serum and propagation in 15% Con A supernatant-containing medium over 90 days. All the experiments were done with the established long-term cultured cell lines. The surface phenotypes of the lines were determined by conventional immunofluorescence microscopy using monoclonal antibodies.

Cell transfer Before passive transfer, cells of the lines (2 x 105 cells/ml) were incubated with MBP (50 # g / m l ) in the presence of syngeneic irradiated (3000 R) spleen cells (1 X 10 7 cells/ml) for 96 h. After incubation the cells were collected and washed twice, and viable cells of various concentrations in a volume of 0.5 ml of phosphate buffer saline (PBS) were injected into the tail vein of recipient mice. In some experiments recipient mice were irradiated (800-1000 R) before cell inoculation.

Clinical assessment The severity of EAE was scored as grade 0-5 according to the following criteria: 0, no abnormalities; 1, decreased tail tone and body tone; 2, a clumsy but otherwise normal gait; 3, definite weakness of one or more limbs; 4, a paraplegic or monoplegic state; 5, a premorbid state. The recipient mice were observed daily for the development of EAE. Animals that did not show any signs of EAE were killed for histologic examination after 2 - 4 weeks.

Histologic examination All animals were assessed histologically by a conventional method using 10% formalin-fixed and paraffin-embedded tissues. Some tissues were fixed with glutaraldehyde and embedded in Epon. Paraffin sections were stained with hematoxylin and eosin. One ~tm Epon sections were stained with toluidine blue and thin sections were stained with uranyl acetate and lead citrate.

Assays of proliferative response Proliferative response was assessed by [3H]thymidine incorporation. Forty thousand line cells per fiat-bottomed microtiter well were cultured in the supplemented RPMI 1640 containing 1% fresh normal mouse serum described above, in the presence of 1 x 106 irradiated (3000 R) syngeneic spleen cells and one of the antigens. In inhibition experiments a monoclonal antibody was simultaneously

284 a d d e d to the culture. The cultures were i n c u b a t e d for 4 d a y s at 37°C in a h u m i d i f i e d 5% C O 2 air a t m o s p h e r e . One /zCi of tritiated t h y m i d i n e ( N e w E n g l a n d N u c l e a r Corp., Boston, M A , U.S.A.) was a d d e d to each well 16 h before harvest with a s e m i - a u t o m a t i c cell harvester. T h e glass fiber disks were c o u n t e d in a P a c k a r d T r i c a r b liquid scintillation counter. D e t e r m i n a t i o n s were d o n e in triplicate a n d the d a t a were expressed as the difference between a n t i g e n - s t i m u l a t e d a n d n o n - s t i m u lated responses (A cpm). T h e s t a n d a r d errors (SE) for the A c p m values were d e r i v e d from the square r o o t of the s u m of the square of the s t a n d a r d errors of the m e a n a n t i g e n - s t i m u l a t e d and n o n - s t i m u l a t e d responses. F o r in vitro s t u d y of genetic restriction of the lines the spleen cells of various strains of mice were used as above.

Monoclonal antibodies A n t i - I - A k (specificity Ia.2), -Thy-1, -Ly-1 a n d -Ly-2 m o n o c l o n a l a n t i b o d i e s were p u r c h a s e d from C e d e r l a n e L a b o r a t o r i e s , O n t a r i o , C a n a d a . A n t i - I - As m o n o c l o n a l a n t i b o d y (specificity Ia.17) was k i n d l y d o n a t e d b y Dr. L. S t e i n m a n ( S t a n f o r d University, Stanford, C A , U.S.A.). W e used a n t i - I - A k which does not cross-react with a n t i - I - As m o n o c l o n a l a n t i b o d y .

Results

General properties of the cell lines T h e long-term cultured cells were f o u n d to b e a r a surface p h e n o t y p e of T h y - l + , L y - I + , 2 -, b y the indirect i m m u n o f l u o r e s c e n c e studies. T h e y r e s p o n d e d well to b o v i n e MBP, guinea pig M B P a n d r a b b i t M B P as well as rat M B P in the presence of A P C , but neither purified p r o t e i n derivative of tuberculin ( P P D ) n o r b o v i n e P L P ( T a b l e 1). Three million of cells of thd lines r e p r o d u c i b l y i n d u c e d clinical a n d histological signs of E A E in naive SJL recipients b e g i n n i n g on d a y 7, without the aid TABLE 1 ANTIGEN SPECIFICITY OF THE ENCEPHALITOGENIC T-CELL LINES Antigen (~g/ml) "

Rat MBP Bovine MBP Guinea pig MBP Rabbit MBP PPD Bovine PLP

Proliferative response to antigens

(50) (50) (50) (50) (50) (100)

Acpm + SE b

SIc

149354+ 4292 101984 + 13 827 120701 + 3 327 119186+ 5868 90+ 63 32+ 67

243.4 166.5 196.9 193.4 1.1 1.0

" Encephalitogenic T cells (4x104) were cultured in 0.2 ml of medium in the presence of 2×106 irradiated (3000 R) SJL spleen cells and the indicated doses of antigens for 96 h. b Stimulation with antigens in culture is expressed as cpm [3H]TdR incorporated as described in Materials and Methods. c Stimulation index (SI) = antigen-stimulated cpm/non-stimulated cpm.

285 TABLE 2 ADOPTIVE TRANSFER OF EAE IN SJL MICE BY THE ENCEPHALITOGENIC T-CELL LINES WITH OR WITOUT MBP STIMULATION AND ITS RADIOSENSITIVITY No. cells transferred a (xlO 6)

Clinical EAE Incidence

3

9/9

1

4/9

0.5 3 irradiated d 30 non-stimulated c

0/4 0/4 0/4

Severity (mean + SE)

Histologic incidence

4.1 + 0.7 3.2 + 0.9 0 0 0

8/8 4/9 0/4 0/4 0/6

Cells of the lines (2 x 105/ml) were stimulated with rat MBP (50 #g/ml) in the presence of irradiated (3000 R) SJL spleen cells (1 × 107/ml) for 96 h or were not stimulated, and transferred i.v. into normal SJL mice. b Number of mice showing signs of EAE/total number of mice in each group. c Animals were graded on a scale of increasing severity, 0-5, as described in Materials and Methods. a MBP-stimulated cells of the lines were irradiated (350 R) before inoculation. c Cells of the lines without stimulation with MBP were transferred i.v. to normal SJL mice. of pertussinogen. However, when 1 x 106 cells were transferred, the disease was less r e p r o d u c i b l e a n d milder. T h e cells i r r a d i a t e d (350 R) before transfer or without M B P s t i m u l a t i o n failed to transfer E A E (Table 2). T h e most p r o m i n e n t histological finding in the recipient was m a r k e d infiltration of n u m e r o u s l y m p h o c y t e s , m a c r o p h a g e s , some p o l y m o r p h o n u c l e a r leukocytes a n d o t h e r cells. T h e lesion was f o u n d in the entire CNS, b u t it was most severe in the lower spinal c o r d (Fig. 1). It was located perivascularly a n d subpially, a n d a l m o s t entirely restricted to the C N S . A x o n s were also d a m a g e d in the severe lesions. D e m y e l i n a t e d axons were observed frequently by electron microscopy.

In vitro genetic restriction T o d e t e r m i n e the r e q u i r e m e n t of Ia antigens for the in vitro proliferative response to MBP, 7 i n b r e d strains of mice were used as A P C d o n o r s (Table 3). T h e i r r a d i a t e d (3 000 R) spleen cells of syngeneic SJL mice p r e s e n t e d M B P to the lines. T h e lines also r e s p o n d e d well to M B P p r e s e n t e d b y A . S W / S n , b u t not b y C 3 H / H e , B A L B / c o r A / J spleen cells. These d a t a indicate that the cell lines are restricted to the H-2 ~ h a p l o t y p e . F u r t h e r m o r e , cultivation with M B P a n d A P C derived from A . S W / S n , b u t not from A . T L mice resulted in proliferation, i n d i c a t i n g that the relevant genes m a p to the fight of the K region of the M H C . Spleen cells from D D D / 1 mice f u n c t i o n e d as A P C in this system, i n d i c a t i n g that the D region of the M H C was irrelevant. Thus, these strains conclusively localize the restriction of the T-cell lines in the I region of the M H C . A d d i t i o n a l i n f o r m a t i o n was gained b y a n t i b o d y - b l o c k i n g e x p e r i m e n t s (Table 4). T h e in vitro proliferative response p r e s e n t e d by A P C from SJL mice was inhibited b y a n t i - I - As m o n o c l o n a l a n t i b o d y , b u t n o t b y a n t i - I - A k. Similar i n h i b i t i o n b y a n t i - I - A~ a n t i b o d y was o b s e r v e d b y using A P C from A . S W / S n or D D D / 1 mice.

286

Fig. 1. Photomicrograph of the T-cell line-induced EAE. SJL mice received 2 - 3 x 106 cells of the T-cell lines, a: This is to illustrate a small power view of the spinal cord of a mouse which showed complete paraplegia on day 8 and was killed on day 17. The lesion is located subpially in the entire lumbar cord. Paraffin section, HE stain, ×65. b: This is to illustrate a higher power view of the dorsal column of thoracic cord obtained from a mouse which showed complete paraplegia on day 13 and was killed on day 23. The infiltrating cells were composed of lymphocytes, macrophages, some polymorphonuclear leukocytes and other cells. Myelinated fibers were severely decreased. Electron-microscopically demyelinated axons were identified frequently. Epon-embedded section, toluidine blue stain, × 300.

287 TABLE 3 G E N E T I C RESTRICTIONS OF A N T I G E N PRESENTATION TO THE ENCEPHALITOGENIC T-CELL LINES IN VITRO " 1rradlated spleen cells b

H-2 alleles K

SJL BA L B / c C3H/He A/J A.SW/Sn A.TL DDD/1

s d k k s s s

Proliferative response to antigen

I

D

Aa

A#

E/~

E.

s d k k s k s

s d k k s k s

(s d k k (s k (s) f

s) r d k k s) t k ?

A cpm 4- SE d

s d k d s d ~

SI ~

152642+5403 - 1702 + 4 844 1201+9983 - 7 4 8 + 306 129187+5389 -1004- 636 837824-3938

175.8 0.8 1.1 0.4 127.6 1.0 47.7

a Cells of the lines (4× 104) were cultured in 0.2 ml of medium with or without rat MBP (10 #g) for 96 h. b Two million irradiated (3000 R) spleen cells from the strains of mice indicated were added at the beginning of the culture period. c Non-s. D region of D D D / I mice was determined by Masuko et al. (1983). d Results are expressed as cpm of [3H]TdR incorporation as described in Materials and Methods. c Stimulation Index as in Table 1. t It is known that H-2' mice do not express both I-E alpha and beta chains (Steinmetz et al. 1982).

These data indicate that the proliferative response of the established T-cell lines are controlled by genes mapping culturing with APC A.SW/Sn

in t h e I - A s u b r e g i o n . A l l o - r e a c t i v i t y w a s o b s e r v e d in

from C3H/He,

BALB/c,

but not the APC

from A/J,

A.TL,

or DDD/1.

In oioo genetic restriction I n o r d e r to see whether the encephalitogenic

a c t i v i t y o f t h e e s t a b l i s h e d cell l i n e s

r e q u i r e s g e n e t i c r e s t r i c t i o n s i m i l a r t o t h a t i n t h e in v i t r o p r o l i f e r a t i v e r e s p o n s e , t h e

TABLE 4 BLOCKING OF IN VITRO A N T I G E N - I N D U C E D PROLIFERATIVE RESPONSE OF THE ENCEPHALITOGEN1C T-CELL LINES BY MONOCLONAL ANTIBODY IN THE PRESENCE OF APC FROM D I F F E R E N T STRAINS OF MICE Monoclonal antibody a Anti-I-As (1/70) Anti-l-A k (1/70)

Proliferative response to antigen" (A cpm + SE) ' SJL

DDD/1

A.SW/Sn

149 354 + 4 292 -364+ 99 122636 + 4299

25 050 + 1935 -1913+1427 18496 + 4 573

129187 + 5 389 525+ 708 113 973 + 2000

a Monoclonal antibody was added at the beginning of the culture period at the final concentration shown. b Cells of lines (4 × 104) were cultured in 0.2 ml of medium with or without rat MBP (10 #g) for 96 h in the presence of 2 x 106 irradiated (3000 R) spleen cells obtained from the indicated strain. ': cpm[~H]TdR incorporated as described m Materials and Methods.

288 TABLE 5 GENETIC RESTRICTION OF IN VIVO ENCEPHALITOGENIC ACTIVITY OF THE T-CELL LINES Recipients ~

SJL C3H/He BALB/c A/J A.SW/Sn A.SW/Sn A.TL DDD/I DDD/1

No. cells transferred ( × 10 6) b

Clinical EAE Incidence ¢

3 10 10 10 10 5 10 10 5

6/6 0/5 0/7 0/4 6/6 0/4 0/4 5/5 0/5

Severity d (mean + SD) 4.6 _+0.5 0 0 0 3.3 + 1.5 0 0 3.6 +_1.5 0

Histological EAE 5/5 0/5 0/7 0/4 4/4 0/4 0/4 3/3 0/4

•' ,MI recipients received 800-1000 R of irradiation immediately before cell inoculation. h All recipients recewed viable cells of the lines after incubation with rat MBP in the presence of irradiated (3000 R) SJL spleen cells for 96 h. " Number of mice showing signs of EAE/total number of mice in each group. d Mean scores (see footnote ¢ to Table 2).

following e x p e r i m e n t was p e r f o r m e d . T h e established T-cell lines were c u l t u r e d with M B P in the presence of syngeneic A P C for 4 d a y s a n d transferred i.v. to the s a m e strains of mice as used in the in vitro e x p e r i m e n t . F o r i m m u n o c o m p r o m i s i n g , these recipients received w h o l e - b o d y i r r a d i a t i o n ( 8 0 0 - 1 0 0 0 R) before inoculation. These recipients d i d not show any signs of graft-vs-host reaction. T h e results are shown in T a b l e 5. T h r e e million cells of the lines were able to transfer E A E to i r r a d i a t e d syngeneic recipients, but not to a n y o t h e r strains of mice. However, 1 × 107 cells i n d u c e d E A E in all A . S W / S n a n d D D D / 1 recipients, b u t failed to transfer E A E to all the other strains of mice. Thus the e n c e p h a l i t o g e n i c T-cell lines were c a p a b l e of i n d u c i n g E A E only when the recipient shared the I region of M H C with SJL, that is, the c o m p l e x of antigen a n d the gene p r o d u c t of I region is the target structure for the lines. These d a t a indicate that the p a t t e r n of genetic restriction required for the in vivo e n c e p h a l i t o g e n i c activity was the s a m e as for the in vitro proliferative responses.

Discussion The present s t u d y was a i m e d at d e t e r m i n i n g the functional role of the Ia antigen for encephalitogenic T l y m p h o c y t e s at the target level of EAE. T h e e n c e p h a l i t o g e n i c T-cell lines, which were established from SJL mice b y a l o n g - t e r m culture, b e a r a Lyt 1 + , 2 - p h e n o t y p e as was r e p o r t e d previously b y o t h e r investigators (Pettinelli a n d M c F a r l i n 1981; B e r n a r d and M a c k a y 1983; L a n d o a n d B e n - N u n 1984) a n d res p o n d e d well to not only rat MBP, which was used for selection of the cell lines, b u t

289 also to bovine, guinea pig or rabbit MBP in the presence of APC. In the in vitro experiments we showed that APC which share the gene products of the I region with SJL mice are capable of presenting the antigen to the cell lines and extended the genetic restriction of cellular interactions between T cells and APC by using monoclonal anti-I-A" antibody. The blocking of antigen-induced proliferative response by anti-I-A ~ antibody showed that these cells must recognize the specific antigen in association with the gene product of I-A subregion of the MHC. Furthermore, we showed that the same pattern of genetic restriction holds for the in vivo encephalitogenic activity of the cell lines as in the in vitro proliferative response. These results indicate that encephalitogenic T cells require recognition of MBP in combination with the Ia antigen at the target level of EAE. In the rat it has been demonstrated that adoptive transfer of EAE by direct or short-term cultured cell transfer is MHC-restricted (Levine et al. 1967; Wegmann and Hinrichs 1984). We have confirmed their observations in mice and, in addition, we indicated the crucial role of the I region within the MHC. In the in vitro experiment the T-cell lines showed allo-reactivity to APC derived from certain strains (e.g. BALB/c, C 3 H / H e ) but not to others (A/J, A.TL, D D D / 1 ) . The reason for the diversity of allo-reactivity is not clear, but it is probable that the allo-reactivity may be directed against non-MHC products such as Mls as previously reported (Molnar-Kimber and Sprent 1980) a n d / o r that these cells may respond specifically to certain haplotypes of allo-antigen. The detail must be awaited for the analysis at the clonal level. The existence of the Ia antigen in the brain is still controversial (Hart and Fabre 1981; Ting et al. 1981). However, in recent studies evidence for its existence has been accumulated (Fontana et al. 1984; Wong et al. 1984). Sobel et al. (1984) demonstrated enhanced staining of the Ia antigen in the brain microvasculature including the endothelium in acute EAE of the guinea pig during preinflammatory and inflammatory periods. Wekerle (1984) observed Ia expression within perivascular infiltrates in rat EAE. We observed similar findings in the brain and spinal cord of EAE-affected mice, while in naive animals we could not find the expression of Ia antigens in the brain (manuscript in preparation). The functional roles of the enhanced expression of the Ia antigen of the brain cells remain obscure, but these observations suggest that activated encephalitogenic T cells might induce or enhance the expression of Ia antigens on certain cells in the brain, thereby the recognition of the target and induction of EAE by encephalitogenic T cells might be facilitated. The precise process in the effector phase of EAE and the regulatory role of brain Ia antigens are yet to be determined. Experiments are now underway to clarify these problems by using the cell lines.

Acknowledgements The authors wish to thank Drs. M.W. Kies, L. Steinman and K. Yamanouchi for their kindness in supplying materials. We also thank Miss Yuki Tachikawa for secretarial assistance, and Dr. Mary Louise Robbins for editing this manuscript.

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