consequences of cytotoxic t lymphocyte interaction with - copy.pdf

8/14/2019 Consequences of Cytotoxic T Lymphocyte Interaction with - Copy.pdf

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C o n s e q u e n c e s o f C y t o t o x i c T L y m p h o c y t e I n t e r a c t i o n w i t hM a j o r H i s t o c o m p a t i b i l i ty C o m p l e x C la ss I - e x p r e s s in gN e u r o n s I n V i v oBy Glenn E Rall Lennart M ucke and M ichael B. A. O ldstoneFrom The Scr ipps Research Ins t itute Div is ion of V irology Department of NeuropharmacologyLa J o lla Ca l i fo r n ia 92 03 7

ummaryN e u ro n s h a v e e v o lv e d s t r a t e g ie s to e v a d e immu n e s u rv e i l l a n c e th a t i n c lu d e a n in a b i l i t y to s y n -th e s i ze th e h e a v y c h a in o f t h e c la ss I ma jo r h i s to c o mp a t ib i l i t y c o mp le x (M H C ) , p ro te in s th a ta re n e ce s s a ry fo r c y to to x ic T ly m p h o c y te (C T L ) re c o g n i t io n o f t a rg e t c el ls . M u l t ip l e v i ru s e sh a v e t a k e n a d v a n t a g e o f t h e l a c k o f C T L - m e d i a t e d r e c o g n i t i o n a n d k i l l in g o f n e u r o n s b y e s t a b -l i s h in g p e r si s t e n t n e u ro n a l i n fe c t io n s a n d th e r e b y e s c a p in g a t t a c k b y a n t iv i r al C T L . W e h a v ee x p re s s e d a cl as s I M H C mo le c u le (D b ) in n e u ro n s o f t r a n s g e n ic mic e u s in g th e n e u ro n -s p e c i f i ce n o la s e ( N S E ) p r o m o t e r t o d e t e r m i n e t h e p a t h o g e n i c c o n s eq u e n c e s o f C T L r e c o g n i t i o n o f v i -r a ll y i n f e ct e d , M H C - e x p r e s s i n g c e n t ra l n e r v o u s s y s t e m ( C N S ) n e u r o n s . T h e N S E - D b t r a ns -g e n e w a s e x p r e s se d in H - 2 b f o u n d e r m i c e , a n d t r a n s g e n e - d e r iv e d m e s s e n g e r R N A w a s d e -t e c t e d b y re v e r s e t r a n s c r ip t a s e -p o ly m e ra s e c h a in r e a c t io n in t r a n s g e n ic b ra in s f ro m s e v e ral li n e s.P u r i fi e d p r i m a r y n e u r o n s f r o m t r an s g e ni c b u t n o t f r o m n o n t r a n s g e n i c m i c e a d h e r e d t o c o v e r -s li ps c o a t e d w i t h a c o n f o r m a t i o n - d e p e n d e n t m o n o c l o n a l a n t i b o d y d i r e c t e d a g a in s t th e D b m o l -e c u le a n d p r e s e n t e d v i r al p e p t i d e t o C T L i n a n M H C - r e s t r i c t e d m a n n e r , i n d i c at i n g t h a t t h e D bmo le c u le w a s e x p re s s e d o n t r a n s g e n ic n e u ro n s in a fu n c t io n a l fo rm . T ra n s g e n ic mic e in fe c t e dw i t h t h e n e u r o t r o p i c l y m p h o c y t i c c h o r i o m e n i n g i t i s v i r u s ( L C M V ) a n d g i v e n a n t i - L C M V ,M H C - r e s t r i c t e d C T L d i s p la y e d a h i g h m o r b i d i t y a n d m o r t a l i t y w h e n c o m p a r e d w i t h c o n t r o l sr e c e i v i n g M H C - m i s m a t c h e d C T L o r e x p r e s s i n g a l t e r n a t i v e t r a n s g e n e s , A f t e r C T L t r a n s f e r ,t r a n s g e n ic b ra in s s h o w e d a n in c re a s e d n u tn b e r o f C D 8 + ce ll s c o m p a re d w i th n o n t ra n s g e n icc o n t ro l s a s w e l l a s a n in c re a s e d ra t e o f c l e a ra n c e o f in fe c t io u s v i ru s f ro m th e C N S . A d d i t io n a l ly ,a n in c re a se in b lo o d -b ra in b a r r i e r p e rme a b i l i t y w a s d e te c t e d d u r in g v i r a l cl e a ra n c e in N S E -D bt ra n s g e n ic mic e a n d l a s t e d s e ve ra l mo n th s a f t e r c l e a ra n c e o f v iru s f ro m n e u ro n s . In c o n t ra s t ,L C M V - i n f e c t e d , n o n t r a n s g e n i c l i t t e r m a t e s a n d m i c e e x p r e s s i n g o t h e r g e n e p r o d u c t s f r o m t h eN S E p r o m o t e r s h o w e d n o C N S d is ea se , n o i n c r ea s e d i n t r a p a r e n c h y m a l C T L a n d n o b l o o d -b ra in b a r r i e r d a m a g e a f t e r th e a d o p t iv e t r a n s fe r o f a n tiv i r a l C T L . O u r s tu d y in d ic at e s th a t v i ra li n f e ct i o n s a n d C T L - C N S i n t er a c ti o n s m a y i n d u c e b l o o d - b r a i n b a r r i e r d i s ru p t io n s a n d n e u r o -lo g ic d is e as e b y a " h i t - a n d - ru n " m e c h a n i s m , t r ig g e r in g a c a s c ad e o f p a th o g e n ic e v e n t s th a t p r o -c e e d s in th e a b s e n c e o f c o n t in u a l v i r a l s timu la t io n .

e v e ra l p ro p e r t i e s u n iq u e to th e c e n t ra l n e rv o u s s y s t e mCNS) 1 l e d t o t h e c o n c e p t t h a t t h e C N S i s a n i m m u n e -

priv i leged s i te (1 , 2 ) . To recognize and lyse ta rge t ce l lsw i t h i n t h e b r a i n p a r e n c h y m a , i m m u n e e f f e c t o r c el ls m u s tc ro ss th e b lo o d -b ra in b a r r i e r , a t i g h t ly p a c k e d w a l l o f e n -d o th e l i a th a t s e p a ra t e s th e c i r c u la to ry s y s t e m f ro m C N Sc e l l s . Wh i l e n u t r i e n t s a n d o x y g e n e i th e r d i f fu s e o r a ret r a n s p o r t e d a c ro s s th e b a r r i e r , t h e p a s s a g e o f b lo o d -b o rn e

Abbreviations used in this paper: aa, amin o acid; CN S, cen tral nervous sys-tem; LCM V, lymph ocytic choriomeningitis virus; mR NA , messenger1KNA; NP, nucleoprotein; NSE , neuron-specificenolase.

p ro te ins and ce l ls ( inc lud ing red b lood ce l ls and res t ingly mp h o c y te s ) i n to th e b ra in i s r e s t r ic t e d , b e c a u s e o f b o thth e f ig h t j u n c t io n s b e tw e e n th e c a p i l l a ry e n d o th e l i a a n d al a c k o f a c t iv e c el l t r a n s p o r t b y th e e n d o th e l i a l c e ll s t h e m -s e lv e s ( fo r r e v ie w s e e r efe re n c e s 3 a n d 4 ) . H o w e v e r , t h eb a r r i er d o e s n o t i s ol a te t h e p a r e n c h y m a f r o m a c t iv a t e d i m -m u n e c e ll s; a c ti v a t ed T l y m p h o c y t e s a n d m o n o c y t e / m a c -ro p h a g e s t r a v e r s e th e in t a c t b lo o d -b ra in b a r r i e r a n d t r a f f i cth ro u g h th e b ra in (5 -9 ) . T h e s e in f i l tr a t in g , a c t iv a t e d T c el lsa re r e s t r i c t e d f ro m re c o g n iz in g C N S t a rg e t s , h o w e v e r , a tl e a s t p ar t ly b e c a u s e o f a p a u c i ty o f c e ll s u r fac e mo le c u le s r e -q u i re d fo r T c e l l - t a rg e t i n t e ra c t io n . R e s id e n t c e ll s o f t h eC N S n o r m a l l y e x p r e ss l o w t o u n d e t e c t a b le l e v e ls o f b o t h

1201 j. Exp. Med. 9 Th e Rockefeller University Press 9 0 0 2 2 - 1 0 0 7 / 9 5 / 1 1 / 1 2 0 1 / 1 2 $2.00Volume 182 N ovem ber 1995 1201-1212


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c la ss I a n d I I M H C m o l e c u l e s ( 1 0 - 1 2 ) . T h e c la ss I M H Cg l y c o p r o t e i n s p r e s e n t f o r e i g n ( v i ra l) p r o t e i n s i n t h e f o r m o f8 - 1 1 a m i n o a c i d (a a) p e p t i d e s t o t h e T c e l l r e c e p t o r o fC D 8 + c y t o t o x i c T l y m p h o c y t e s . N e u r o n s , b o t h i n v i v o( 1 3 - 1 5 ) a n d i n v i t r o (1 2 , 14 , 1 6 - 1 8 ) , d o n o t e x p r e ss M H Cc la s s I m o l e c u l e s a n d , h e n c e , d o n o t s e r v e a s t a r g e t s f o rC T L - m e d i a t e d l y si s. M u l t i p l e R N A a n d D N A v ir u se s , i n -c l u d in g m e m b e r s o f th e Herpes - , P aramyxo- , R habdo- , P i -coma- , a n d Arenavirus f a m i li e s , i n f e c t n e u r o n s o f t h e C N S( f or r e v i e w s e e r e f e re n c e s 1 9 - 2 1 ) . V i r u s e s f r o m e a c h o ft h e s e f a m i l i e s a r e a b l e t o e s t a b l i s h l a t e n t o r p e r s i s t e n t i n f e c -t i o n s i n C N S n e u r o n s , m o s t l i k e l y b e c a u se v i r a l p e r s i s te n c ei n n e u r o n s t h r o u g h i m m u n e e v a s i o n is p r e f e r a b l e t o l ys is b ya n t i v i r a l C T L o f t h e s e e s s e n t i a l a n d i r r e p l a c e a b l e c e l ls .

E a r li e r st u di es b e g a n t o c h a r a c t er i z e n e u r o n - v i r u s - C T Li n t e r a c t io n s u s i n g t h e O B L - 2 1 n e u r o n a l c e l l l i n e ( 14 ).T h e s e c e ll s c o n t a i n n e u r o f i l a m e n t s a n d s h o w e l e c t r o p h y s i -o l o g i c r e s p o ns e s c o n s i s t e n t w i t h n e u r o n s ( 22 ) , a l t h o u g h ,u n l i k e C N S n e u r o n s , t h e y a r e d i v i d i n g ce l ls . L i k e n e u r o n si n v i v o , O B L - 2 1 c e ll s f ai l t o e x p r e ss M H C m o l e c u l e s , a n d ,a l t h o u g h i n f e c t e d w i t h v i r u s a n d e x p r e s s i n g v ir a l p r o te i n so n t h e i r s u r fa c e s , w e r e n o t l y s e d b y a n t i v i r a l C T L ( 1 4) .H o w e v e r , a ft er c o m p l e m e n t a t i o n o f c la ss I M H C a n t i g e n -p r e s e n t i n g c a p a c i t y b y t r a n s f e c t io n w i t h a D b - e n c o d i n g r e t -r o v i r a l v e c t o r , i n f e c t e d O B L - 2 1 c e ll s w e r e l y s e d b y a n t i v i -r a l C T L ( 1 4) .

I n t h i s p a p e r w e e x t e n d t h e s e i n v i t r o o b s e r v a t i o n s i nv i v o . F i r s t , t r a n s g e n i c m i c e t h a t e x p r e s s a cl as s I M H C m o l -e c u le o n C N S n e u r o n s w e r e e s ta b li s he d . S e c o n d , M H C -e x p r e s s in g n e u r o n s p u r i f i e d f r o m s u c h t r a n s g e n i c m i c ew e r e s u s c e p t ib l e t o l ys is b y v i r u s - s p e c i fi c M H C - r e s t r i c t e dC T L , w h e r e a s n e u r o n s f r o m n o n t r a n s g e n i c m i c e w e r e n o t .C N S d i se a se w a s i n d u c e d i n v i r a ll y i n f e c t e d t r a n s g en i c m i c ea f t er a d o p t i v e t r a n s f e r o f v i ru s - s p e c i f i c , M H C - r e s t r i c t e dC T L . T h e d i se a s e l a s te d f o r s e ve r a l m o n t h s a f te r C T Lt r a ns f e r a n d w a s a s s o c ia t e d w i t h a n i n c r e a s e d n u m b e r o f i n -t r a p a r e n c h y m a l C D 8 + c e l ls a n d a c h r o n i c d e s t a b i l iz a t i o n o ft h e b l o o d - b r a i n b a r r i e r .

a t e ri a ls a n d e t h o d sAnimals, Cells, and Viruses. C 5 7 B L / 6 ( H - 2 b ), SJ L ( H - 2 9 , a n d

B A L B / c B y J ( H - 2 a ) i n b r e d m i c e w e r e o b t a i n e d f r o m t h e c l o s e dbree d ing fac i l i ty o f Th e Scr ipps Rese arch Ins t i tu te (La JoUa , CA) .A n i m a l s w e r e m a i n t a i n e d i n c o n d i t i o n s c o n s is t e n t w i t h A m e r i c a nA s s o c i a ti o n fo r t h e A c c r e d i t a t i o n o f L a b o r a t o r y A n i m a l C a r e r e g -u la t ions th rou gho u t the cou rse o f the inves t iga t ion .L y m p h o c y t i c c h o r i o m e n i n g i t i s v i r u s ( L C M V ) s t ra i n A r m -s t r o n g C A 1 3 7 1 c l o n e 5 3 b ( L C M V A R M ) w a s u s e d i n t h e s es tud ies. The o r ig in , c lon in g and p laque pur i f ica t ion o f s tock v i rush a v e b e e n r e p o r t e d ( 2 3 , 2 4 ) . V i r u s w a s g r o w n i n b a b y h a m s t e rk idney ce l l s and was p laque pur i f ied and t i te red on Vero ce l lmon o layers a s desc r ibed (24). BAL B c lone 7 (H-2 a ) f ib rob las tsa n d V e r o c e l l s w e r e g r o w n i n M E M s u p p l e m e n t e d w i t h 1 0h e a t - i n a c t i v a t e d F C S , 1 m M r - g l u t a m i n e , 1 04 U / m l p e n i c i ll i n ,a n d 1 0 m g / m l s t r e p t o m y c i n . M C 5 7 ( H - 2 b ) f i b ro b l a st s w e r e c u l -t u r e d i n R P M I 1 6 40 w i t h t h e s u p p l e m e n t s l i s te d a b o v e . C T Lc l o n e s r e st r i c te d b y H - 2 b ( c l on e N P 1 8 r e c o g n i z i n g n u c l e o p r o t e i n[ N P ] a a r e s id u e s 3 9 6 - 4 0 4 ; 2 5 ) a n d H - 2 a ( c lo n e H D 8 r e c o g n i z i n g

NP aa res idues 118-127 ; 26 ) were used . These c lones were m ain -ra ined in cu l tu re as desc r ibed (25) .P e r s i st e n t in f e c t i o n o f m i c e w a s a c h i e v e d b y i n o c u l a t i o n o f1 ,0 00 P F U o f L C M V A R M i n tr a c er e b ra l ly i n t o n e w b o r n m i c ewi th in 18 h o f b i r th . Such m ice ca r ry in fec t ious v irus and v i ra lnuc le ic ac id in many ce l ls , inc lud in g neurons , th roug hou t the i rlives (27).Cloning Procedures and Establishment of Transgenic M ice. Stan-dard mole cu la r techn iques (28, 29 ) were used fo r the con s t ruc -t i o n o f t h e N S E - D b t r a n sg e n e . T h e N S E e x p r e s si o n v e c t o r ( 3 0 ,31) was ob ta ined f rom S . Forss -Pe t te r , (The Scr ipps Research In -s t i tu te ) and the M o/ D b c lone (32) f rom R . F lave l l , (Ya le Uni ver -s i ty , N e w H a v e n , C T ) . P r o m o t e r - t r a n s g e n e j u n c t i o n s c r e a t e d b yt h e s u b c l o n i n g w e r e s e q u e n c e d b e f o r e m i c r o i n j e c t i o n . F e r t i l i z e do o c y t e s w e r e o b t a i n e d f r o m ( C 5 7 B 1 / 6 S J L) f e m a l e m i c e . P u r i -f i c a ti o n o f t h e t r a ns g e n e , p r e p a r a t i o n o f m i c e , m i c r o i n j e c t i o n , a n de m b r y o i m p l a n t a t io n s w e r e c a r r i ed o u t a s d e s cr i b e d ( 3 3 - 3 5 ) .T r a n s g e n i c m i c e w e r e i d e n t i f i ed b y t a i l b i o p s y a n d s u b s e q u e n tD N A iso la t ion . 10 FLg o f DN A was s lo t b lo t te d on to f il te r s (Ny -t r an ; S c h l e i c h e r & S c h u e l l , I n c ., K e e n e , N H ) a n d p r o b e d w i t ht h e S V 4 0 s e q u e n c e l o c a t e d a t t h e 3 ' e n d o f th e N S E e x p r e ss i o ncassette.R everse Transcr ip tase -P CR R T -P C R ) . B r a i n s a n d o t h e r o r -g a ns w e r e r e m o v e d f r o m s a l i n e- p e r f u se d m i c e a n d w e r e s na p f r o -zen in l iqu id n i t rogen . The t i ssues were s to red a t -7 0~ be fo reh o m o g e n i z a t i o n . T i s s u e s w e r e h o m o g e n i z e d u s i n g a V i r t i s h e a r(Vir t i s Co . , Inc . , Gard ine r , NY) fo r 30 s in 1 ml T r i - reagen t /100m g t is su e ( M o l e c u l a r R e s e a r c h C e n t e r , I n c . , C i n c i n n a t i , O H ) ,a n d R N A w a s p ur i fi e d a cc o r d in g t o t h e ir r e c o m m e n d e d p r o t o -c o l . 5 00 n g o f t o ta l R N A w a s r e v e rs e t r a ns c r i b e d b y t h e r a n d o mp r i m i n g m e t h o d , u s i n g r a n d o m h e x a m e r s ( P h a r m a c i a B i o t e c h ,Inc . , P isca taway , NJ) and reverse t ransc r ip tase (GIBCO BRL,G a i t he r sb u r g , M D ) . T h e R N A - c D N A d u p l e x w a s t h e n s u b-j e c t e d t o 4 0 r o u n d s o f P C R ( 60 ~ a n n e a l i n g t e m p e r a t u r e ; T a q Ip o l y m e r a s e f r o m G I B C O B R L ) , u s i n g 2 0 - m e r o l i g o n u c l e o t i d epr imers des ign ed to ampl i fy t ransgene spec i f ic sequences : A = 5 'G A G A T C G A C T C T A G A G G A T C 3 '; B = 5 ' G C G C TC T G G T T G T A G T A G C C 3 ' . T h e P C R p r od u c ts w e r e v is ua l-i z e d b y e t h i d i u m b r o m i d e i n t e r c al a t i o n o n a 1 a g a ro s e g e l.Hippocampal Neur on Cultures and Protein Detec tion. E m b r y o n i cm i c e ( d a y 1 6 -1 8 ) w e r e u s e d f o r t h e p r e p a r a t i o n o f d is s o c ia t e dh i p p o c a m p a l n e u r o n c u l t ur e s f o l lo w i n g t h e p r o t o c o l o f B a n k e rand Gos l in (36) wi th minor modif ica t ions (37 ; Ra l l , G. F . , un -pub l i shed resul ts ) . B r ie f ly , h ipp ocam pi were d issec ted f rom em -b r y o s a n d p l a c e d i n i c e - c o l d p l a t i n g m e d i u m ( D M E M s u p p l e -m e n t e d w i t h 1 0 f e ta l b o v i n e s e r u m , 1 04 U / m l p e n i c i l l in , a n d 1 0m g / m l s t r e p t o m y c i n ) . T h e t is su es w e r e w a s h e d t h r e e t i m e s w i t hi c e - c o l d P B S a n d d i g e s te d w i t h t r y p s i n - E D T A ( S ig m a C h e m i c a lCo . , S t . Lou is , M O) a t 37~ fo r 15 ra in . The t i ssue was washedt w i c e w i t h p l a t in g m e d i u m , m e c h a n i c a l l y d is s o c i at e d w i t h f i r e -po l i shed p ipe t te s , and passed th rough a ce l l s t ra ine r (Fa lcon Lab-ware , Ox nard , CA) to ob ta in a s ing le -ce l l suspension. The neuronswer e spun a t 150 g fo r 7 ra in on to a se rum cush ion , re suspended ,c o u n t e d , a n d p l a t e d o n t o e i t h e r p o l y - L - ly s i n e ( S i gm a C h e m i c a lC o . ) o r a n t i b o d y - c o a t e d , a c i d - w a s h e d , r o u n d g l a s s c o v e rs l ip s( C a r o l in a B i o l o g ic a l S u p p l y C o . , B u r l i n g t o n , N C ) a t a d e n s i ty o f5 0 0 c e l l s / m m2.

Ind iv idua l covers l ips were incub a ted w i th a f f in i ty -pur i f ied goa tan t i -mouse IgG (Cappe l Labora to r ies , Cochranv i l le , PA; 10 p~g /s l ip ) fo r 4 h , washed wi th PBS and then coa ted wi th rnAbs spe -c i f ic fo r e i the r the D b c lass I M H C mole cu le (B22 . 249 .R1 , 1 :500 ;C e d a r l a n e L a b o r a to r i e s L t d ., H o m b y , O n t a r i o , C a n a d a ) o r t o t h eLa c lass I M H C mo lecu le (30-5 -7S , 1 :500 ; Cedar lane Lab ora to -

1 2 0 2 C T L C la ss I M H C - e x p r e s s i n g N e u r o n In t e ra c t io n s I n V i v o


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r i e s L td . ) i n doses o f 3 Fg /cov er s l i p ( 38 , 39 ) . Cover s l i p s wer ep l a c ed i n a h u m i d i f ie d i n c u b a t o r o v e r n i g h t , a n d , i m m e d i a t e l y b e -f o r e n e u r o n p l a t i n g , t h e a n t i b o d y w a s a s p i r a te d a n d t h e s li ps w e r ew a s h e d o n c e w i t h P B S , p H 7 .4 .

4 h a f t e r t he ne u r o ns wer e adde d to t he cover s l i p s , t he s li p sw e r e p l a c e d o n t o p o f a c o n f l u e n t p r i m a r y a s t r o c y t e f e e d e r l a y e r( 40 ), i n s e r u m - f r ee n e u r o b a s al m e d i u m ( G I B C O B R L ) s u p p l e -m e n t e d w i t h g r o w t h f a c t o r s ( B 2 7 a d d i t i v e ; G I B C O B R L ) . T h ec el ls w e r e m a i n t a i n e d a t 3 7 ~ i n 5 C O 2 .

In Vitro Kill ing Assay. N e u r o n s c u l t u r e d i n v i t r o f o r 4 8 hw e r e e i t h e r u n t r e a t e d o r w e r e c o a t e d w i t h p e p t i d e s r e p r e s e n t i n gL C M V C T L e p i to p e s r e s tr ic t e d b y e i t h er H - 2 b ( N P a a 3 9 6 - 4 0 4 :N H 2 - F Q P Q N G Q F I - C O O H ) o r H - 2 d ( N P aa 1 1 8- 12 7 : N H 2 -S E R P Q A S G V G - C O O H ) a t a c o n c e n t ra t i o n o f 4 0 be g/ 2 1 04t a rg e t s 1 h b e f o r e t h e a d d i t i o n o f C T L . H - 2 b - o r H - 2 a - r e s t r i c t e dC T L c l o n e s w e r e t h e n a d d e d t o t h e c u l t u r e s a t a 1 : 1 r a t i o , a n dt h e y w e r e a l l o w e d t o i n c u b a t e f o r 6 h . A f t e r r e m o v a l o f th ec l o n e s a n d d e b r i s b y s u c c e ss i v e P B S w a s h e s , t h e n e u r o n s w e r ef i x e d w i t h 2 p a r a f o r m a l d e h y d e a n d w e r e c o u n t e r s t a i n e d w i t hh e m a t o x y l i n . 1 0 r a n d o m f ie ld s w e r e c o u n t e d p e r s l i de , a n d t h er es u lt s w e r e c o n f i r m e d i n t h r e e i n d e p e n d e n t e x p e r i m e n ts . M C 5 7( H- 2 b ) and BA L B c17 ( H - 2 a ) fi b r ob las t s se r ved as con t r o l s .A dop t ive Transfe r o f M em ory C TL in t o P ersi s ten t ly In fec tedMice . A d o p t i v e t r a n s f e r o f a n t i v i r a l C T L i n t o p e r s i s t e n t l y i n -f e c t e d m i c e h a s b e e n d e s c r i b e d ( 4 1 ) . T h e m e m o r y c y t o t o x i c e f -f e c t o r c el ls t r a n s fe r r e d w e r e o b t a i n e d f r o m n o r m a l a d u l t C 5 7 B I / 6o r S J L m i c e i n f e c t e d 6 0 - 1 2 0 d e a r l i er b y i n t r a p e r i t o n e a l i n o c u l a -t i o n o f 2 X 1 0 s P F U o f L C M V . S p l ee n s w e r e r e m o v e d a n d d i s so -c i a te d , a n d a s i n g l e - c e ll s u s p e n s i o n w a s p r e p a r e d a n d c u l t u r e d i n2 4 - w e l l p l a te s w i t h s y n g e n e i c L C M V - i n f e c t e d a n d i rr a d ia t e dp e r i t o n e a l m a c r o p h a g e s (a s s t i m u l at o r s ) a n d s y n g e n e i c i r r a d i a t e dsp l een ce i ls ( a s f eede r ce ll s ). Ad he r en t l ymp hoc y tes ( secondar i es )w e r e e x p a n d e d t h r e e t i m e s o n i n f e c t e d , i r r a d i a te d s t i m u l a t o r c e ll sb e f o r e t r an s f e r i n t o a g e - m a t c h e d ( 6 - 1 3 - w k - o l d ) p e r s i s te n t l y i n -f e c t e d m i c e a n d n o n t r a n s g e n i c c o n t r o l s .

T r a n s g e n i c a n d n o n t r a n s g e n i c m i c e ( H - 2 b) w e r e b r e d w i t hn o n t r a n s g e n i c S JL m i c e ( H - 2 s) t o g e n e r a t e H - 2 bxs m i c e f o r t h ea d o p t i v e t r a n s f e r e x p e r i m e n t s . T h e p r o g e n y o f s u c h b r e e d i n g sw a s i n fe c t e d w i t h L C M V ( 1 03 P F U ) w i t h i n 2 4 h o f b ir t h t o e s -t a b l is h p e r s i s te n t i n f e c t io n s . T h e p r e s e n c e o f v i r u s i n t h e s e m i c e( b e t w e e n 4 a n d 8 w k ) w a s d e t e r m i n e d b e f o r e th e a d o p t i v e t r a n s -f e r by se r um p la que assay. 24 h be f o r e t h e t r ansf e r o f 5 X 106 sec -o n d a r y C T L i n t r a p e r i t o n e a l l y , m i c e w e r e i r r a d i a te d w i t h 4 0 0 r a dt o a c c o m m o d a t e t h e a d o p t i v e l y t ra n s f e r re d c el ls . T h e h e a l t h o ft h e a n i m a l s w a s m o n i t o r e d d a i l y f r o m 1 to 3 0 d a f t e r a d o p t i v et r a ns f e r a n d w e e k l y t h e r e a ft e r , a n d e v a l u a t e d o n a f o u r - l e v e l s c al e:1 , h e a l t h y a n d i n d i s t i n g u i s h a b l e f r o m u n i n f e c t e d n o r m a l m i c e ; 2 ,f u r s l i gh t ly r u f f l ed , pos tu r e hu nch ed , eyes pa r t ia l l y c losed , r e -d u c e d m o b i l i t y ; 3 , r uf f le d , h u n c h e d , a n d t r e m u l o u s , e y e s m u c o u sf i ll e d a n d c l o se d , s i g n i f i c a n t m o t o r i m p a i r m e n t ; 4 , d e a d .

V i r a l ti t er s i n s e r u m w e r e m o n i t o r e d 6 , 1 5 , 6 0 , a n d 1 2 0 d a f t e ra d o p t i v e t r a n s fe r , a n d m i c e f r o m e x p e r i m e n t a l a n d c o n t r o l g r o u p swer e k i l l ed a t 15 and 60 d a f t e r adop t ive t r ansf e r f o r h i s to log i ca ls tud i es .Detection of CD 8 Lymphocytes within Brain Sections. /Mice wer ed e e p l y a n e s t h e t iz e d w i t h a 3 . 8 s o l u t i o n o f c h l o r a l h y d r a t e a n dw e r e p e r f u s e d w i t h n o r m a l s a l i n e. B r a i n s w e r e r a p i d l y r e m o v e d ,q u i c k f r o z e n i n a d r y i c e / is o p e n t a n e b a t h , a n d s t o r e d a t - 7 0 ~1 0 - 1a m h o r i z o n t a l s e c t io n s w e r e c u t a n d fi x e d i n 9 5 e t h a n o l f o r1 5 m i n a t - 2 0 ~ w e r e b l o c k e d w i t h a v i d i n - b i o t in ( V e c to r L a b -o r a t o ri e s , I n c . , B u r l i n g a m e , C A ) , a n d w e r e i n c u b a t e d f o r 1 .5 hw i t h m A b s t o t h e m u r i n e C D 8 m o l e c u l e ( C D 8 a a n d L y 3 , d il u t e d1:5 ; P h a r m i n g e n , S a n D i e g o , C A ) . A f t e r w a s h i n g , s e c t i o n s w e r e

i n c u b a t e d w i t h b i o t i n y l a t e d s e c o n d a r y a n t i b o d i e s , f o l l o w e d b y a na v i d i n - b i o t i n re a c t i o n ( A B C E l i t e ; V e c t o r L a b o r a t o r ie s , I n c . ) a n da c o l o r r e a c t io n u s i n g d i a m i n o b e n z i d e n e a n d h y d r o g e n p e r o x id e .S e c t io n s w e r e l i g h tl y c o u n t e r s ta i n e d w i t h h e m a t o x y l i n a n d w e r em o u n t e d w i t h A q u a m o u n t ( L e m e r La b o ra to r ie s , N e w H a v e n , C T ) .

T h e m e d i a n v a l u e s o f t h e n u m b e r o f C D 8 + l y m p h o c y t e s p e rb r a i n s e c t i o n w i t h i n a g i v e n g r o u p w e r e c a l c u l a t e d . A t l e a s t f o u rh o r i z o n t a l s e c t i o n s w e r e e v a l u a t e d p e r m o u s e b r a i n .Blood-Brain Barrier Permeabili ty . M i c e w e r e d e e p l y a n e s t h e -t i z e d w i t h a 3 . 8 s o l u t i o n o f c h l o r a l h y d r a t e . 2 m l o f a 2 E v a n sb l u e s o l u t i o n i n P B S w a s i n f u s e d tr a n s c a r d ia l l y o v e r a l - r a i n p e -r i o d . 3 ra i n l a te r , m i c e w e r e p e r f u s e d w i t h 4 p a r a f o r m a l d e h y d ei n P B S , a n d b r a i n s w e r e r e m o v e d a n d s to r e d i n 0. 2 p a r a f o r m a l -d e h y d e i n P B S .

R e s u l t sThe Class I M H C Molecu le , D ~ , I s E xpressed in Neuro ns o f

Transgenic Mice. T r a n s g e n i c m i c e w i t h n e u r o n a l e x p re s -s i o n o f t h e c la ss I M H C m o l e c u l e D b w e r e e s t a b l is h e d b yi n j e c ti o n o f o oc y te s w i t h a m i n i g e n e c o n t a i n i n g t h e c o m -p l e te D b c o d i n g s e q u e n c e u n d e r t h e c o n t r o l o f t h e N S Ep r o m o t e r ( F ig . 1 ). T h e D b m i n i g e n e ( 4 0) w a s c l o n e d i n t ot h e B a m H I r e s tr i c ti o n s it e w i t h i n e x o n 2 o f t h e N S E c a s-s e t te ( F ig . 1 ), a n d t h e r e s u l t i n g p l a s m i d w a s c h a r a c t e r i z e db y r e s t r ic t i o n a n al ys is a n d s e q u e n c i n g o f p r o m o t e r - t r a n s -g e n e j u n c t i o n s . T h e l i ne a r iz e d , g e l - p u r if i e d N S E - D b t r a ns -g e n e w a s t h e n m i c r o i n j e c t e d i n t o ~ 2 0 0 ( C 5 7 B 1 / 6 S JL )F 2o n e - c e l l s t ag e e m b r y o s .

F r o m t h is m i c r o i n j e c t i o n , 4 2 m i c e w e r e b o r n , 1 2 o fw h i c h c o n t a i n e d t h e t r a n s g e n e . F i v e o f t h e se f o u n d e r m i c ew e r e s e l ec t e d f o r b r e e d i n g a n d w e r e b a c k - c r o s s e d o n t o t h eC 5 7 B 1 / 6 b a c k g r o u n d . A f t e r t h r e e b ac k - cr o s se s , t h e C T Lr e s p o n s e w i t h i n t h e s e m i c e w a s c o m p l e t e l y r e s t r ic t e d to t h eH - 2 b h a p l o t y p e a s d e t e r m i n e d b y c h r o m i u m r e le a se a s sa ys( d a t a n o t s h o w n ) .

T i s s u e e x p r e s s io n o f t h e N S E - D b m e s s e n g e r R N A( m R N A ) w a s a n a ly z e d b y R T - P C R o n R N A i so la te df r o m t r a n s g e n ic a n d n o n t r a n s g e n i c m o u s e t is su e s ( b ra i n ,

el e2 . ,~1I 5 REGULATORYEQ~.CE ~ ~ NSE CASSETTE

l k b ~ poly ~I , t o b M I N , Q E N E

el I1 e 2 i 2 e 3 - 8 3 U T R5 0 0 b p

Figure 1. Structure of the NSE -Db fusion gene. The NSE -D b con-st ruct consists of 2 .8 kb ofS ' NSE regulatory sequence, NSE exon 1 an dintron 1, and 15 bp ofN SE exon 2 plus polyl inker . The D b minigen e in-cludes all 8 exons a nd the f i rst two int rons of the mu r ine D b gene. Poly-adenylation signals are provided b y 3' untranslated sequences (UT R) ofthe D b minigen e. SV40 sequence at the 3 ' end of the construct faci li tatesidenti f icat ion of t ransgenic mice by slot blot analysis ofge nom ic DN A. e,exon; i , in t ron.1 2 0 3 R a l l e t a l.


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Figure 2. ExpressionofDb mRN A in brains oftransgenic mice. Brainswere removed from transgenic (+) and nontransgenic (- ) mice. TotalRN A was extracted and subjected to R T- PC R as described in Materialsand M ethods, Primers A and B were used to amplify transgene-specificmR NA. The primer pair was designed to differentiate spliced and un-spliced templates by size; the primers span intron 1 o f the D b sequence, sothat the predicted PCR product derived from spliced mRNA is 180 bpsmaller than the product derived from either unspliced RN A or residualDNA in the preparation (Fig. 2 13 .Spliced mR NA (as a 320-bp PCRproduct) was detected in each of the five transgenic lines, and, in somecases, a PC R product corresponding o D NA or unspliced RN A (500 bp)was seen (lines 23 and 29). N o PC R product was amphfied in nontrans-genic mice (line 21-). Results obtained in three mice per group areshown.

spleen, kidney, pancreas, l iver, testes, gut, thymus, lung,hear t , and opt ic and sc ia t ic nerves) . Tota l bra in RNA iso-lated fro m mice of six transgenic lines (7, 9, 11, 21, 23, and2 9 ) c o n ta in e d t r a n sg e n e -d e r iv e d mRN A , w h e re a s b ra inRNA from nontransgenic l i t te rmates d id not (Fig . 2 A).Transgenic l ines 9 (da ta not shown) and 11 were used forsu b se q ue n t e x p e r imen t s . N o sp li c ed N S E-D b m RN A w a sde tec ted in o ther organs except for in the testes of line 29(da ta not shown). Line 29 was not used for fur ther exper i -ments .

To ta l RN A p u r i f i e d f ro m p r ima ry a s t ro c y te /mic ro g l i a lcu l tures der ived from t ransgenic n eona ta l m ice o f l ines 9a n d 1 1 w e re a lso a n a ly z e d b y th e R T- PC R a p p ro a ch . N oR N A P C R p r o d u c t c o r r e sp o n d i ng t o t h e N S E - D b t ra n s-gene was de tec ted in these cul tures (da ta not sho wn).

Th e NS E- D b expression a t the pro te in leve l was too lo wto b e d e t e c t e d b y immu n o s t a in in g o f ti ssue se c tio ns . H o w -ever , neuro na l expression of D b pro te in was demo nstra tedb y c u l tu r in g p r ima ry e mb ry o n ic h ip p o c a mp a l n e u ro n s a n dtesting their abili ty to grow on glass coverslips coated withan mA b speci f ic for e i ther D b or for a he te ro lo gous M H Cmolecule (ant i -Ld) . Neu ron s f rom t ransgenic (Db-express-ing) mice adhered to an t i -D b- , but not to a n t i -L &coa tedcoversl ips (Fig . 3) . Nontransgenic neurons d id not a t tachto e i ther type o f coverslip , confi rm ing the absence ofMHC c lass I on the i r surface . Notably , the ant ibody used ,B.22 .249, i s confo rma t ion depende nt (32) . Therefo re , the

D b molecule i s expressed in a conform at iona l ly correc tform on the surface of t ransgenic neurons.

The Transgene-derived D b Molecule Conferred C T L Rec-ognition and Lysability to CNS Neurons In Vitro. We n e x t d e -te rmined whether t ransgenic neurons could present an t igenand se rve as ta rge ts for CTL-media ted lysis in a neurona lsurvival assay. Primary n euro ns d o no t take up S~Cr; ther e-fore, instead o f standard c hr om iu m release assays, an alter-na t ive assay was used . Transgenic and nontransgenic neu-rons were incuba ted wi th pept ides encoding LCMV-spec if icepi topes tha t a re rest r ic ted by e i ther H -2 b or H -2 d (25 , 26 ,35) . Pept ide-coa ted neurons were reac ted wi th spec i f ica n t i -L CM V H -2 b - o r H -2 &re s t r i c t e d C TL c lon e s, a n d then u mb e r o f vi a bl e n e u ro n s w a s d e t e rmin e d a f t er a 6 -h i n c u -b a t io n . D b -e x p re ss in g n e u ro n s i n c u b a te d w i th H -2 b -rest r ic ted v i ra l pept ide and a matched CTL c lone showed asignificant red ucti on (73 ) i n cell viabili ty (P < 0.05),whereas nontransgenic neurons d id not (Fig . 4) . These da tawere reproduc ib le in three independent exper iments .

Persistently Infected, Transgenic Mice Developed C N S Diseaseafter Adopt ive Transfer of LCM V-sp ecif ic Db-restricted CT L.NSE-Db-expressing mice were mainta ined in a spec i f icpa thogen-free fac i l i ty . No d i ffe rences in appearance , fecun-di ty , or l i fe span were noted be tween uninfec ted t ransgenicanimals and nontransgenic l i t te rmate contro ls (da ta notshown).

To de te rmine the ant igen-present ing capac i ty of t rans-genic neurons in v ivo and the consequ ences o f the i r in te r-ac t ion wi th Db-rest r ic ted CTL, mice were infec ted as neo-nates with LCMV. In this persistent infection, most t issues,inc luding the CNS, l iver , sp leen , and k idney, conta inLCMV ; w i th in t h e CN S, t h e n e u ro n i s t h e o n ly c e l l t y p einfec ted (42, 43). Adopt ive t ransfe r of v i rus-spec i f ic,MH C-re s t r i c t e d CTL (4 1 , 4 4 ) r e su l t s i n CTL-me d ia t e dc learance of LC M V from a ll s ites , o ther than neu rons andrena l g lomerul i , wi th in 10-30 d a f te r t ransfe r . The eventua le l imina t ion o f v i rus and v ira l nuc le ic ac id f rom neurons oc-curs over a longer per iod (60-120 d a f te r adopt ive t ransfe r)and does not in volve obv ious inf i l t rat ion of the bra in pa-rench yma (41) . The mech anism of v i ral c lea rance f romnontransgenic , persisten t ly infec ted bra ins is not co mple te lyunderstood, but does not seem to occur v ia c lassica l CTL-target cell interactions.

N S E- D b t r an sg e nic mic e , N SE- a my lo id p re c u r so r p ro -te in (APP) t ransgenic mice (expressing APP as a non-MHCtransgenic contro l ; 31) , and nontransgenic l i t te rmates werein fec t e d w i th L CM V a t b i r th . Ea c h o f th e se g ro u p s o f m ic ewas genera ted by breedin g an H- 2 b t ransgenic parent wi tha nontran sgen ic SJL (H-2 9 mou se, resulting in H- 2 bX~ off-spr ing . Secon dary im mu ne T lymph ocytes ra ised in H-2 bor H -2 ~ mice w ere then adopt ive ly t ransfe rred in to the per-sistently infected H- 2 bx~ animals. Th is expe rim ent was basedon the fo l lowing ra t iona le : both H- 2 b and H- 2 ~ ant i -LCMV CTL could recognize v i ra l ly infec ted ce l ls in pe-ripheral t issues o f persistently infecte d H- 2 bx~ mice, buto n ly H -2 b a n t i -L CM V CT L w o u ld r e c o g n iz e vi ra l a nt ig e nc o mp le x e d w i th t h e D b mo le c u le o n N S E-D b tr a n sg e n icneurons. Thus, i f mice beca me sick or d ied as a conse-

1204 CTL Class I MHC -expressing Neuron Interactions In Vivo


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Figure 3. Expressionof D b molecules on the surface of n eurons isolated from the CNS of transgenic mice. Primary embryonic hippocampal neuronsfrom transgenic and nontransgenic litters were plated onto coverslips coated with mAbs to either D b (B22.249) or La (30-5-7S) as described in Materialsand M ethods. Photographs were taken 48 h after plating. Staining with antibody against the neuronal marker, microtubule-associatedprotein 2 indicatedthat >95 of the adherent cells were neurons.

que nc e o f CT L in t era c tions w i th t he D b e xp re ssed o n ne u -rons, th is should be seen only in NS E- D b t ransgenic m icerece iv ing the H-2b-rest r ic ted CTL (Fig . 5) .

A n ima l he a l th w a s mon i to re d da i ly a nd inde pe nde n t lyby a t least two invest iga tors a f te r adopt ive t ransfe r of CT L.The ge ne t ic p ro f il e o f the mic e a nd the CT L tha t t hey r e -ce ived were unknown to the observers to avoid b ias. Per-sis tently infec ted mice appeared hea l thy before the adopt ivetransfer. 5-15 d after transfer, when CTL are kill ing virallyinfected MHC-expressing cells in peripheral t issues (liver,sp leen , k idney, e tc .) (41) , some mice showed mild to mod-erate i l lness (clinical levels 2-3) in all experimental groups.Th e com bine d resul ts (Table 1) of three independ ent ad op-t ive t ransfe r exper iments using a mi nim um of four mice perexper imenta l group revea led the fo l lowing: (a) NSE -D btransgenic mice exposed to H -2 b CT L sho wed signif icantlyh ighe r morb id i ty a nd mor t a l i t y t ha n N SE-A PP o r non -t ransgenic contro ls rece iv ing simi la r immunotherapy; (b)N S E- D b mic e ha d a h igher m orb id i ty a nd m or t a li t y w he ng i v en H - 2 b C T L c o m p a r e d w i t h H - 2 + C T L , w h e re a s H - 2 band H -2 s CT L had a s imi la r e ffect in the co ntro l groups;and (c) NS E- D b mic e th at surv ived th e initial 1 5-d viralc learance per iod remained sick throughout the observa t ion

120 5 R.all et al.

per iod (c l in ica l s tages 2-3) , whereas contro l mice re turnedto clinical stage 1 after viral clearance.NS E- lY Transgenic Mice Receiving Adoptive C T L TherapyShowed Increased Numbers of CD 8 + Lymphocytes in the C N SParenchyma and Accelerated Viral Clearance. Brains were re -mo ved from mice a t var ious t ime points a f te r adopt ive t rans-fe r a nd s t a ine d w i th mA bs to mur ine CD 8 to c ompa re t henum be r o f in f il t ra t ing C TL w i th in t he b ra in s o f N SE- D btransgenic versus contro l mice . Photo micro graph s of repre-senta t ive rout ine bra ins a re shown in Fig . 6 , and a quant i ta -t i ve summ a ry i s show n in Ta b le 2 . U n in fe c t e d N S E- D btransgenic bra ins conta ined ra re CTL (Fig . 6 A; Table 2 ,group A), suggest ing tha t the expression of D b on neuronsper se is not sufficient to recruit T cells into the paren-c hyma . Th e CD 8 CT L found w i th in t he b rain s o f pe rs i s -t e n tly i n fec t e d mic e w e re d i s tr i but e d un i fo rmly th rou ghou tthe parenchyma, and both t ransgenic and nontransgenicmice k i l led a t var ious ages a f te r neona ta l LCMV infec t ionc on ta ine d s im i la r numbe rs o f i n t r a pa re nc hyma l CD 8 Tcells (Fig. 6 B; Table 2, groups B and C). In contrast, 15 daf ter adopt ive t ransfer of Db-rest r ic ted ant i -L CM V C TL ,there was a marked increase in the num ber of CD 8 + Tcel ls wi th in bra ins of persis tent ly infec ted , NS E- D b trans-


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Figure 4. Lysisof Db-expressing primary neurons coated with appro-priate viral peptide by Db-restricted LCMV-specific CTL. Primary neu-rons were incubated for 1 h with peptides encoding epitopes for LCMVrestricted by either H-2b (viral NP, aa 396-404) or H -2 d (NP, aa 11 8-127) or with no pe ptide (- -). One hour after peptide addition, H -2b- orH-2a-restricted antiviral CT L were added to the cultures. 5-6 h later,neurons were fixed, stained, and counted as described in Materials andMethods. N umb ers of neurons in cultures incubated w ith no peptide andH-2 d CTL w ere arbitrarily defined as 100 . Data points represent thenumb er of remaining neurons expressed as a percentage of this control;error bars indicate SD. Similar results we re obtained indep endently in twoadditional experiments (data not shown). * P values


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Fig ure 6 . Increased numb er of CD8 + cells in brains of t ransgenic, pers is tent ly infected mice af ter adoptive t ransfer of H-2b-res tr ic ted ant i-L CMVCTL. S ec t io n s o f cau d a te A-D) or cerebel lum (E) were s tained for the presence ofC D8 lymph ocytes as descr ibed in Mater ials and Methods , and werecounters tained with h ematoxylin . (A) Uninfected N SE- D b transgenic mouse , no CT L given; (B) pers is tently infected NSE -D b mouse , no CT L given;(C) pers is tent ly infected , NS E-D b t ransgenic mouse, day 15 af ter adoptive t ransfer of H-2 b secondaries ; (D) pers is tent ly infected , nontransg enic mous e,day 15 af ter adoptive t ransfer of H -2 b secondaries ; (E) pers is tent ly infected , NS E-D b t ransgenic mouse, day 40 af ter adoptive t ransfer of H-2 b secondar ies . 100.

o

~

LEVl~LOF

NSE-Db CONTROL NSE-D CONT ROL NSE-D CONTROLD A Y 0 D A Y 1 5 D A Y 4 0

Fig ure 7 . Clearance of infect ious v irus f rom brains of transgenic andcontrol m ice. Brains of pers is tently infected N SE- D b or control (non-transgenic or NSE-APP) mice were removed 15 or 40 d af ter adoptivetransfer of H-2 b CTL, weighed, homog enized , and analyzed for the pres -ence of infect ious v irus by plaque assay. At leas t four animals per groupwere evaluated. Resul ts represent means + SD.

F ig u r e 8 . A b n o r mal p e rmeab i l ity o f th e b lo o d - b r a in b a rr i er in pe r si s -tent ly infected NS E-D b mice af ter adoptive t ransfer of H-2 b LCM V-sp e-

cific CTL. Pers is tent ly infected mice were in jected with Evans blue andpeffused as descr ibed in Mater ials and Methods . (A) Nontransgenicmouse, no CT L given; (B) pers is tent ly infected NSE -D b transgenicmou se given H-2 ant iv iral CTL 120 d previous ly; (C) pers is tent ly in-fected, non transgenic mous e given H- 2 b CTL and sho wing s igns of s ick-ness after 15 d after transfer; (D and E) persiste ntly infected NS E- D b mic egiven H-2 b CT L 120 d previous ly.

1 2 0 7 Ra l l e t a l.


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w o u l d b e a s s o ci a te d w i t h a n a l t e r a ti o n o f t he b l o o d - b r a i nb a r r i e r , m i c e w e r e i n j e c t e d w i t h E v a n s b l u e , a d y e w h i c hb i n d s t o s e r u m p r o t e i n s a n d i s e x c l u de d f r o m t h e b r a i n p a -r e n c h y m a b y t h e n o r m a l b l o o d - b r a i n b a r r i e r . I n p e r s i s -t e n t l y i n fe c t e d N S E - D b t r a ns g e n i c m i c e , l e a k a g e o f t h e d y ei n t o t h e b r a i n p a r e n c h y m a w a s o b s e r v e d a s e a r l y a s 1 5 dn = 4 ) data no t show n) an d as la te as 120 d n = 5 ) af ter

ad o p t iv e t r an s fe r o f H -2 b r e s t r i c t ed C T L F ig . 8 ; T ab le 3 ) .T h e s e r e s u l t s i n d i c a t e a p r o l o n g e d a b n o r m a l p e r m e a b i l i t yo f th e b l o o d - b r a i n b a r r i e r, w h i c h p e r s is t e d in t h e a b s e n c eo f v i r a l an t i g en . S u b t l e d i f f e r en ces i n t h e p a t t e r n o f p e r m e-ab i l i t y ch an g es co u ld b e r e l a t ed t o v a r i a t i o n s i n t h e d i s t r i -b u t io n o r ac t i v i t y o f t h e i n f i lt r a t i n g l y m p h o cy te s .

B y c o n t r a st , n o o t h e r g r o u p o f m i c e , i n c lu d i n g t r a n -s i e nt l y si c k no n t r a n s g e n ic m i c e k i ll e d 9 d a f t e r C T L a d m i n -i s t r a t i o n , s h o w e d a n y a b n o r m a l i t y i n t h e p e r m e a b i l i t y o fth e b lo o d -b ra in b a r r i e r T ab le 3; Fig . 8 ).

i s c u s s i o nT h e c u r r e n t s t u d y r e v e a l e d a n u m b e r o f n o v e l f in d in g s .

T h e e x p r e s si o n o f c la ss I M H C g l y c o p r o te i n s in n e u r o n s o fN S E - D b t r a n sg e n i c m i c e h a d n o d e l e te r i o u s e f f ec t s o n t h eh e a l th , f e c u n d i t y , o r l if e sp a n o f u n i n f e c t e d m i c e . M H Cc la ss [ - e x p r e s s i n g h i p p o c a m p a l n e u r o n s o f t r a n sg e n i c m i c ep r e s en t e d e x o g e no u s v ir a l p e p t id e t o H - 2 - m a t c h e d C T Lin v i t ro , r e su l t i n g i n n eu ro n a l l y s i s . I n v iv o i n t e r ac t i o n s o fa n t i v i r a l H - 2 b - r e s t r i c t e d C T L w i t h D b - e x p r e s s i n g n e u r o n sin m ice p e r s i s t en t ly i n f ec t ed w i th L C M V resu l t ed i n s ig n i f -i can t m o rb id i t y an d m o r t a l i t y . D i sease i n d u ced in t h e t r an s -g e n i c m i c e w a s a s s o c i a t e d w i t h a n i n c r e a s e i n t h e n u m b e ro f C D 8 T l y m p h o c y t e s w i t h i n t h e b r a i n p a r e n c h y m a , a nin c reased r a t e o f c l ea ran ce o f i n f ec t i o u s v i ru s f ro m in fec t e dn e u r o n s , a n d a n i n c r e a s e d p e r m e a b i l i t y o f t h e b l o o d - b r a i nb a r r i e r t h a t c o n t i n u e d f o r s e v e ra l m o n t h s a f te r t h e a d o p t i v et r an s f e r o f C T L a n d a f t er t h e v i r u s w a s c l e a re d f r o m i n -f e c t e d n e ur o n s . A l t h o u g h v i r u s w a s c l e a r e d f r o m D b - e x p r e s -s in g n eu ro n s b y 1 5 d a f t e r ad o p t iv e t r an s fe r , n o o b v io u s l y -s is o f n eu ro n a l ce ll s w as o b se rv ed .

O u r i n v i t r o f i n d i n g s i n t r a n s g e n i c p r i m a r y e m b r y o n i cn e u r o n s c o n f i r m a n d e x t e n d p r e v i o u s r e s u l t s o b t a i n e d i nO B L -2 1 n eu r o n a l ce l ls 1 4 , 1 7 ). I n th o se s tu d ie s it w assh o w n th a t O B L -2 1 ce l l s w ere u n ab le t o p resen t v i r a l an t i -g e n t o C T L b e c a u s e o f a p a u c i t y o f c la ss I M H C o n t h eO B L - 2 1 c e l l s u rf a c e. M o l e c u l a r c o m p l e m e n t a t i o n w i t h ar e t ro v i r a ll y e x p r es s e d cl as s I M H C m o l e c u l e m a d e i n f e c te dO B L -2 1 ce ll s r eco g n izab l e an d ly sab le b y an t iv i r a l C T L .A l th o u g h O B L -2 1 ce ll s sh a re m a n y ch a rac t e r is t i c s w i thn eu ro n a l ce ll s e . g ., ex p res s io n o fn e u ro f i l a m e n t s , N S E , an dv o l t a g e -d ep e n d e n t p o t a s s iu m ch an n e l s ; 2 2) , t h ey , u n l ik ep r im ary n eu ro n s , a r e d iv id in g , t r an s fo rm ed ce l l s t h a t g ro wi n t h e p r e s e n c e o f se r u m .

E x p res s in g D b M H C c la s s I p ro t e in s i n n eu ro n a l ce ll s v i at r a ns g e n i c t e c h n o l o g y a n d p a n n i n g w i t h m A b t o D b i n t h ecu r r en t s t u d y a l l o w ed th e i n v i t ro ana ly s is o f p r im ar y n eu -r o n a l c e l l - v i r u s - C T L i n te r a c t io n s . A s th e s e p r i m a r y n e u -ro n s d o n o t d iv id e i n cu l tu re an d a r e g ro w n in t h e ab sen ceo f s e r u m , t h e y r e s e m b l e d i f f e r e n ti a t ed n e u r o n s i n t h e i n t a c t

C N S m o r e c l o s e ly t h a n O B L - 2 1 c el ls . W h e n t r a ns g e n icD b - e x p r e s s i n g p r i m a r y n e u r o n s w e r e p u l s e d w i t h v i r a l p e p -t i d e o r i n f ec t ed w i th v i ru s R a i l , G . , an d M . B . A . O ld -s t o n e , u n p u b l is h e d r e su lt s) a n d c h a l l e n g e d w i t h M H C -m a t c h e d a n t i v i r a l C T L , t h e y w e r e r e c o g n i z e d a n d l y s e d ,w h e r e a s p r i m a r y n e u r o n s f r o m n o n t r a n s g e n i c m i c e u n d e rs im i l a r co n d i t i o n s w e re n o t . T h u s , ex p res s io n o f a cl as s IM H C m o l e c u l e o n p r i m a r y n e u r o n s i n v i t r o w a s s uf f ic i e ntto r e su l t i n ly si s b y ap p ro p r i a t e C T L .I n t e re s t i ng l y , w e d i d n o t f in d e v i d e n c e o f C T L - m e d i -a t ed l y si s o f n eu ro n s i n b ra in s o f t r an sg en ic m ic e , d esp i t et h e p r e s e n c e o f i n t r a p a r e n c h y m a l C T L a n d r a p i d v ir a lc l e a ra n c e f r o m t h e t r an s g e n ic C N S . H o w e v e r , i t s h o u ld b en o ted t h a t t h i s s t u d y w as n o t d es ig n ed to accu ra t e ly q u an t i -t a t e n e u r o n a l c o u n t s a n d t h a t a s u bt l e d r o p o u t o f n e u r o n sm a y h a v e o c c u r re d . N e v e r t h e l e s s , n e u r o n s o f t h e h i p p o -c a m p u s a n d P u r k i n j e c e ll la y e r o f th e c e r e b e l l u m a r e u n i -f o r m l y i n fe c t e d b y L C M V i n v i v o 4 1, 4 2) ; y e t a t n o t i m eaf t e r ad o p t iv e t r an s fe r , i n n o an im a l , r eg a rd l e s s o f h ea l t h ,w ere m o rp h o lo g ica l ch an g es co n s i s t en t w i th ce l l l y s i s o b -se rv ed in t h ese r eg io n s . T h u s , w h i l e t r an sg en ic n eu ro n s ex -p res s in g t h e co r r ec t v i r a l p ep t id e b o u n d to a tr an sg en icM H C m o l e c u l e w e r e s u i ta b le C T L t a r g e t s i n v it r o , s u c hn eu ro n s d id n o t ap p e a r t o b e e f f ec t i v e ly l y sed i n v iv o . T h i so b s e r v a t i o n m a y b e d u e t o a r e s tr i c te d n u m b e r o f C D 8 C T L w i t h i n t h e b r a i n , w h i c h l i m i t s th e c y t o t o x i c e f f e c t o fth ese ce l ls . W e b e l i ev e t h is p o ss ib i l it y is u n l ik e ly , h o w ev e r ,g i v e n t h e r a p i d r a t e o f v ir a l cl e a r an c e a n d b l o o d - b r a i n b a r -r i e r d am ag e w i th in t r an sg en ic b ra in s .

A l t e r n a ti v e l y, o n e m i g h t c o n s i d e r t ha t , w h i l e t h e s e n e u -ro n s ex p res s t h e t r an sg en e i n v i t ro , t h ey m ay n o t i n v iv o , a sn o d i r e c t e v i d e n c e e .g ., i m m u n o h i s t o c h e m i c a l i d e nt i fi c a -t i o n o f t h e t r an sg en e p ro d u c t i n b ra in sec t i o n s ) is p ro v id ed .W e f ee l t h a t o u r i n ab i l it y t o d e t ec t t h e c la s s l m o le cu l e i nb ra in sec t i o n s w as d u e t o t ech n ica l p ro b l em s r eg a rd in g t h ed i ff i cu l ty o f i m m u n o s t a i n i n g w i t h i n t h e b r a i n , c o u p l e dw i th t h e l ack o f a su i t abl e , h ig h -a f f i n i t y an t ib o d y , an d n o td u e t o t h e a b s e n c e o f e x pr e s s io n i n v i v o . T h e a d o p t i v et r a ns f e r e x p e r i m e n t s p r o v i d e s t r o n g e v i d e n c e f o r f u n c t i o na lp r o t e i n e x p r e s s io n in v i v o : t h r e e g r o u p s o f m i c e N S E - D b ,N S E - A P P , a n d n o n t r a n s g e n i c l i tt e r m a te s ) , t w o l i n e s o f t h eN S E - D b m i c e l in es 9 a n d 1 1) , a n d t w o d i f f e r e n t i m m u n el y m p h o c y t e h a p l o t y p e s b a n d s) w e r e u s e d t o d e m o n s t r a t ea se l ec t i v e e f f ec t o f H -2 b an t iv i r a l l y m p h o cy te s o n v i r a l lyin fec t ed H -2 b t r an sg en ic n eu ro n a l t a rg et s . B ecau se o n ly t h ec o m b i n a t i o n o f m i c e e x p r e s s in g t h e D b m o l e c u l e w i t hM H C - m a t c h e d H - 2 b i m m u n e l y m p h o c y t e s r e su l te d i np a t h o l o gi c a l c on d i t io n s i n c re a s e d i n t r a p a r e n c h y m a l C T L ,i n c re a s e d b l o o d - b r a i n b a r r i e r p e r m e a b i l it y , p r o l o n g e d s i c k -n ess ), f u n c t io n a l ex p res s io n o f t h e c l a ss I M H C t r an sg en e i nn eu ro n s i s t h e m o s t p l au s ib le ex p lan a t io n fo r t h ese d a t a.

H o w t h e s e C T L e x e r t t h e i r v i r u c i d a l , b u t n o t c y t o c i d a l ,e f f ec ts i n v iv o i s n o t k n o w n . I t is co n ce iv ab l e t h a t f ac to r sr e l eased f ro m n eu ro n s o r g l i a l ce l l s i n t e r f e r e w i th l y t i cC T L - n e u r o n a l i n t e r a c t i o n s c o n t r i b u t i n g t o t h e r e l a t i v e l yi m m u n o s u p p r e s s i ve / i m m u n e - p r i v il e g e d m i c r o e n v i r o n m e n to f t h e C N S , a n d t h a t s u ch f a c to r s i n t e rf e r e w i t h t h e C T L -m e d ia t ed cy to ly s is o f v i t a l l y i n f ec t ed n eu ro n s . I so l a t i o n o f

1208 CT L Class I MH C-expressing Neu ron Interactions In Vivo


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ab l e 2. Accumulation of C D 8 + T Cells Present in Brains of Mice Receiving Adoptive Transfer of Immune LymphocytesMedian number ofintraparenchymalAdoptive transfer of CD8 + T cellsH-2 b anti-LCMV CTLGroup Mice Virus Transgene (day observed) Median Range

A 3 - + - 12 (0-17)B 4 + - - 48 (19-82)C 4 + + - 55 (14-89)D 5 + + + (15) 124 (48-211)E 4 + - + (15) 62 (23-77)F 4 + + + (40) 51 (15-69)

*Persistendy infected (groups B-F) or uninfected (group A) NSE=Db transgenic (groups A, C, D, and F) or nontrausgenic (groups B and E) micewere given either H-2b-restricted antiviral CTL (groups D-F) or no CTL (groups A-C). Brains were examined immunohistochemicallyfor thepresence ofCD8 + T cells at either 15 (groups D and E) or 40 (group F) d after transfer as described in Materials and Methods.*Values given represent the median number of CD8 positive cells found/horizontal brain section (determined from four sections per mouse,three-five mice per group).

Table 3. Increase n Transgenic Mouse Blood-Brain Barrier Permeabili ty Is Dictated by Expression of the Transgenein Virally Infected Mice Given Haplotype-matched Antiviral C TLExperimental model Result

Neurons expressing i n vivoMHC class I LCMV

Adoptive transferof Db-restricted,LCMV= specific CTLPercent incident ofincreased blood-brainbarrier permeability

+ + + 100 (5/5)+ - - o 0 / 5 )+ - + 0 (0/3)+ + - 0 (0/4)- + + o 0 / 5 )- + - 0 0 / 5 )

*Persistently nfected (+) or uninfected (- ) NSE-D b transgenic (+) or nontransgenic (-) mice were given Db-restricted antiviral CTL 120 d beforeblood-brain barrier assessment. Permeability was determined by entry of Evans blue into the parenchyma. The percentages of mice with Evans blueinfiltration are given, with the total number of mice per group in parentheses.

neurons from this envir onme nt by in vitro culture may re-move the m f rom these factors and allow lysis to occur.

Expression of a functional MH C molecule on neur onsalso induced a protracted disruption of the blood-brainbarrier. This effect may be mediated by soluble CTL prod-ucts such as cytokines. Since the effector cells in the adop-tive splenocyte transfer have been shown to be virus-spe-c i f ic CD8 + (41), the most likely cytokines would be IFN-~/and TNF-oL. Studies to directly test this hypothesis are cur-rently underway using IFN-'y and TNF-oe knockou t mice.Cytokines have recently been shown to cause direct injurywithin the CNS in which injection (45) or endogenoussynthesis (46) of cytokines resulted in several clinical andhistopathologic changes within the brain, including blo od-

1209 1Kall et al.

brain barrier damage.After adoptive transfer of MHC-restricted virus-specific

CTL into mice whose neurons contain virus but lackexpression of MH C class I molecules, clearance occurs by60- 120 d (41). Interestingly, in the model repo rted here,clearance occurs much earlier, by day 15 (Fig. 7). In bothinstances, clearance of virus from neurons occurs in theabsence oflysis of these cells, suggesting that intrapa renchy -real cytokine production may also participate in the clear-ance of infectious virus from transgenic brains. Inhibitionof viral synthesis in the absence of cell death has bee n do c-umented in the CTL response to hepatitis B virus infectionof hepatocytes (47-49) and in H IV (50). In these systems,inflammatory cytokines such as IFN-~/ and TNF-ci can


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d o w n - r e g u l a t e v i r al e x p r e s s i o n w i t h o u t c o n c o m i t a n t c e lld e a t h .

T h e s t a b il i t y o f t h e b l o o d - b r a i n b a r r i e r i s c r i ti c a l t o t h ee x c l u s i o n o f p o t e n t i a l l y n e u r o t o x i c o r c y t o l y t i c b l o o d -b o r n e m o l e c u l e s ( s u c h a s c y t o k i n e s ) f r o m t h e C N S p a r e n -c h y m a a s w e l l a s t o t h e m a i n t e n a n c e o f a n a p p r o p r i a t e h o -m e o s t a t i c o s m o t i c b a l a n c e w i t h i n t h e b r a i n ( 5 1 ). W h i l er a p i d a lt e r a ti o n s i n t h e p e r m e a b i l i t y o f t h e b l o o d - b r a i n b a r -r i e r m a y r e s u l t i n e d e m a a n d d e a t h ( 5 2 , 5 3 ), s l o w e r c h a n g e si n p e r m e a b i l i t y n e e d n o t b e l if e t h r e a t e n i n g b u t m a y l e a d t om o r e c h r o n i c n e u r o l o g i c s y m p t o m s ( 5 4) . I n a g r e e m e n tw i t h t h is c o n c e p t , w e o b s e r v e d a s i g n i fi c a n t i n c r e as e i n a n -i m a l s i c k n e s s a n d d e a t h a t e a r l y t i m e s a f t e r a d o p t i v e t r a n s -

f e r, w i t h c o n t i n u e d s i c k n es s o f s u rv i v i n g m i c e t h r o u g h o u tt h e 1 5 0 - d o b s e r v a t i o n p e r i o d .

S e v e r a l C N S d i se a se s h a v e b e e n a s s o c ia t e d w i t h t r a n s ie n to r p e r m a n e n t c h a ng e s i n b l o o d - b r a i n b a r r i e r p er m e a b i l i ty ,i n c l u d i n g t h o s e a ss o c i at e d w i t h C N S i n f e c t i o n ( 5 4 - 5 8 ) , d e -m y e l i n a t i n g d i se a s e s ( 5 9 ), a n d t u m o r s ( 6 0 ). O u r s t u d y , i nw h i c h a b n o r m a l p e r m e a b i l i t y o f t h e b l o o d - b r a i n b a r r i e ra n d c l i n i c a l i l ln e s s w e r e d e t e c t e d l o n g a f t e r v i r u s h a d b e e nc l e a r e d f r o m t h e m i c e ( F i g . 7 ) , i n d i c a t e s th a t v i r a l i n f e c t i o n sa n d C T L - C N S i n te r a ct i on s m a y i n d u c e b l o o d - b r a i n b a r-r i e r d i s r u p t io n s a n d n e u r o l o g i c d i se a s e b y a h i t - a n d - r u nm e c h a n i s m , t r i g g e r i n g a c a s c ad e o f p a t h o g e n i c e v e n t s t h atp r o c e e d s i n t h e a b s e n c e o f c o n t i n u a l v i r a l s t i m u l a t i o n .

The au thors acknow ledge the techn ica l a ssi s tance o f Monica Ham balko and L i l iana R iquer , the secre ta r ia la i d o f A m y G o d d a r d , a n d D a v i d B l o o m f o r r e v ie w i n g t h e m a n u s c r ip t .Th is wor k was suppor ted by U.S . Pub l ic Hea l th g ran ts AI09484 and N S12428 , an d by a fe l lowsh ip f rom theJ . D. and Ida Le ipe r Founda t ion and Na t ion a l Ins ti tu tes o f Hea l th t ra in ing g ran t MH191 85 to G. F . Ra l l . L .Mu cke was suppor ted , in pa r t, by a Har ry Weave r Neurosc ience Scho la rsh ip Aw ard f rom the N a t iona l Mul-tiple Sclerosis S ociety.Address co r respondence to Glenn F . Ra i l , The Fox Chase Cancer Cen te r , 7701 Burho lme Avenue , Ph i la -delphia, PA 19111.Received or publication 2 February 1 99 5 and in revised orm 31 M ay 199 5.

e f e r e n c e s1 . M edaw ar , P . 1974. T ransp lan ta t ion b io logy : an appra isa l . Mt.SinaiJ. Med. 4 1 : 7 6 0 - 7 6 4 .2 . Bar t le t t , P .F . , R .S .C . Ker r , and K.A Ba i ley . 1989 . Express iono f m a j o r h i s t o c o m p a t i b i l i t y a n t ig e n s i n t h e c e n t r al n e r v o u s

system. Transplant. Proc. 2 1 : 3 1 6 3 - 3 1 6 5 .3 . G r e e n w o o d , J . 1 9 9 1. M e c h a n i s m s o f b l o o d - b r a i n b a r r i e rb r e a k d o w n . Neuroradiology.3 3 : 9 5 - 1 0 0 .4 . S t re i le in , J .W . 1993. Im m une p r iv i lege as the resu l t o f loca lt i s sue ba r r ie rs and immunosuppress ive mic roenv i ronments .Curr. Opin. Immunol. 5 : 4 2 8 - 4 3 2 .5 . W e k e r l e , H . , C . L i n i n g t o n , H . L a s s m a n n , a n d K . M e y e r -m a n n . 1 9 86 . C e l l u l a r i m m u n e r e a c t i v it y w i t h i n t h e C N S .Trends Neurosci. 9 : 2 7 1 - 2 7 7 .6 . W e k e r l e , H . , D . S u n , R . L . O r o p e z a - W e k e r l e , a n d R . M e y -e rman n . 1987 . Im mu ne reac t iv i ty in the ne rvous sys tem:m o d u l a t i o n o f T l y m p h o c y t e a c t i v a t i o n b y g l i a l c el ls . J. Exp.Biol. 132 :43-57 .7 . Hick ey , W.F . , and H. Kim ura . 1987 . G ra f t ve rsus hos t d iseasee l ic i ts express ion o f c lass I and I I M H C an t igens and p resenceo f s c a tt e r e d T l y m p h o c y t e s i n t h e r a t C N S . Proc. Natl. Acad.Sci . USA. 8 4 : 2 0 8 2 - 2 0 8 6 .8 . H i c k e y , W . F . , B .L . H s u , a n d H . K i m u r a . 1 9 9 1. T l y m p h o -cy te en t ry in to the cen t ra l ne rvous sys tem. J. Neurosci. Res.28:254--260.

9 . Olds tone , M .B .A . , and P. J. Sou the rn . 1993. T ra f f ick ing o fa c t i v a t ed C T L i n t o t h e c e n t r al n e r v o u s s y s te m : u s e o f a t r a n s -g e n i c m o d e l . J . Neuroimmunol. 4 6 : 2 5 - 3 1 .10 . Lam pson , L . 1987 . Mo lecu la r bases o f the im mu ne responseto neu ra l an t igens . Trends Neurosci. 10 :211-216 .

11 . Dre w, P . , M. Lonergan , M . G olds te in , L . Lampso n , K.O z a t o , a n d D . M c F a r l i n . 1 9 93 . R e g u l a t i o n o f M H C c la ss Iand j32 -mic rog lobu l in gene express ion in hum an n eurona lcells. J. Immunol. 1 5 0 : 3 3 0 0 - 3 3 1 0 .

12 . Massa, P .T . , K. O za to , and D .E . M cFar l in . 1993 . Ce l l typespec i fic regu la t ion o f ma jo r h is tocomp at ib i l i ty comp lex (MH C)class I gene ex pression in astrocytes, olig oden drocy tes and n eu -r o n s . G L / A . 8 : 2 0 1 - 2 0 7 .

1 3 . L a w r e n c e , J . M . , R . J . M o r r i s , D J . W i l s o n , a n d G . R a i s m a n .1990. M echan isms o f a l log ra ft re jec t ion in the ra t b ra in . Neu-roscience. 3 7 : 4 3 1 - 4 6 2 .14 . Jo ly , E . , L . M ucke , and M .B .A. O lds tone . 1991. Vira l pe rs i s-t e n c e i n n e u r o n s e x p l a i n e d b y a l a c k o f M H C c la ss I e x p r e s -sion. Science Wash. DC). 2 5 3 : 1 2 8 3 - 1 2 8 5 .15 . Mu cke , L . , and M.B .A. O lds tone . 1992. The express ion o fM H C c lass I an t igens in the b ra in d i ffe rs mark ed ly in acu tea n d p e r s i s t e nt i n f e c ti o n s w i t h l y m p h o c y t i c c h o r i o m e n i n g i t i sv i rus . J . Neuroimmunol. 3 6 : 1 9 3 - 1 9 8 .1 6 . L a m p s o n , L . , a n d C . F i s h e r . 1 9 8 4 . W e a k H L A a n d ~ - 2 m i -c r o g l o b u l i n e x p r e ss i o n o f n e u r o n a l c e ll l in e s c a n b e m o d u -la ted by in te r fe ron . Proc. Natl. Aca d. Sci . USA . 8 1 : 6 4 7 6 -6480.17 . Jo ly , E . , and M.B .A . Old s tone . 1992. N euro na l ce l l s a re de f i -c i e n t i n l o a d i n g p e p ti d e s o n t o M H C c la ss I m o l e c u l e s. Neu-ron. 8 : 1 1 8 5 - 1 1 9 0 .1 8 . K e a n e , R . W . , M . W . T a l l e n t, a n d E . R . P o d a c k . 1 9 92 . R e s i s -tance an d susce p t ib i l i ty o f neura l ce ll s to lys is by cy to tox iclymphocy tes and by cy to ly t ic g ranu les . Transplantation Balti-more). 5 4 : 5 2 0 - 5 2 6 .

1 2 1 0 C T L C la ss I M H C - e x p r e s s i n g N e u r o n I n t e ra c t io n s I n V i v o


11/12

19 . Sch l i t t , M. , R .B . Chr on is te r , and R . J . W hi t ley . 1991 . Pa tho -g e n e si s a n d p a t h o p h y s i o l o g y o f v i r a l i n fe c t i o ns o f t h e c e n t r alne rvous sys tem. In I n f e c ti o n s o f t h e C e n t r a l N e r v o u s S y s t em .W . S c h e l d , e d i t o r . R a v e n P r es s, N e w Y o r k . 7 - 1 8 .20 . Ah m ed , R . , and J .G. S tevens . 1990 . Vira l pe rs i s tence , InF i e l d s V i r o l o g y . B . N . F i e l d s a n d D . M , K n i p e , e d i t o rs .R a v e n P r e s s , N e w Y o r k . 2 4 1 - 2 6 6 .21 . Es i r i , M .M . , a nd P .G .E . K enned y . 1992. V irus d iseases, InG r e e n f i e l d s N e u r o p a t h o l o g y . F i f t h E d . J .H . A d a m s a n d L . W .D u c h e n , e d i t o r s . O x f o r d U n i v e r s i t y P re ss , N e w Y o r k . 3 3 5 -399.22 . Ryder , E .F . , E .Y. Snyder , and C .L . Cepko . 1990 . Es tab l i sh -m e n t a n d c h a r a c t e r i z a ti o n o f m u l t i p o t e n t n e u r a l c e l l li n e s u s -i n g r e t r o v ir u s v e c t o r - m e d i a t e d o n c o g e n e t ra n sf er . J. Neuro-biol. 2 1 : 3 5 6 - 3 7 5 .2 3 . D u t k o , F . J . , a n d M . B . A . O l d s t o n e . 1 9 8 3 . G e n o m i c a n d b i o -l o g i c al v a r i a t io n a m o n g c o m m o n l y u s e d l y m p h o c y t i c c h o r i -ome ning i t i s v i rus s t ra ins . J . Gen. Virol. 6 4 : 1 6 8 9 - 1 6 9 8 .2 4 . O l d s t o n e , M . B . A . , R . A h m e d , M . J . B u c h m e i e r , P . B l o u n t ,and A. T ishon . 1985. Pe r tu rba t ion o f d i f fe ren t ia ted func t ionsd u r i n g v i r a l i n fe c t io n s i n v i v o . I . R e l a t i o n s h i p o f l y m p h o c y t i cc h o r i o m e n i n g i t i s v i ru s a n d h o s t s t ra in s t o g r o w t h h o r m o n edef ic iency . Virology. 142 :158-174 .

2 5 . W h i t t o n , J .L . , J . R . G e b h a r d , H . L e w i c k i , A . T i s h o n , a n dM . B . A . O l d s t o n e . 1 9 8 8. M o l e c u l a r d e f in i t i o n o f a m a j o r c y -t o t o x i c T l y m p h o c y t e e p i t o p e i n t h e g l y c o p r o t e i n o f l y m -p h o c y t i c c h o r i o m e n i n g i t i s v i r u s . J . ViroI. 6 2 : 6 8 7 - 6 9 5 .

2 6 . W h i t t o n , J. L . , A . T i sh o n , H . L e w i c k i , J . G e b h a r d , T . C o o k ,M. S a lva to , E . Jo ly , and M.B .A. O lds tone . 1989. Mo lecu l a ra n al ys es o f a fi v e a m i n o a c i d c y t o t o x i c T l y m p h o c y t e ( C T L )e p i to p e : a n i m m u n o d o m i n a n t r e g i on w h i c h i n d uc e s n o n r e -c ip roca l CTL c ross reac t iv i ty . J . Virol. 6 3 : 4 3 0 3 - 4 3 1 0 .2 7. B u c h m e i e r , M J . , R . M . W e l s h , F J . D u t k o , a n d M . B . A . O l d -s t o n e . 1 9 80 . T h e v i r o l o g y a n d i m m u n o b i o l o g y o f l y m -p h o c y t i c c h o r i o m e n i n g i t i s v i r u s i n f e c t io n . Adv. Immunol . 30:2 7 5 - 3 3 1 .2 8 . A u s u b e l , F . M . , R . B r e n t , a n d R . E . K i n g s t o n . 1 9 8 7 . C u r r e n tP r o t o c o l s i n M o l e c u l a r B i o l o g y . G r e e n e P u b l i s h i n g A s s o c i -a te s a n d W i l e y I n t e r sc i e n c e , N e w Y o r k , N Y . 2 . 0 . 1 - 3 .1 9 . 8 .29 . Sam brook , J . , E .F . F r i t sch , and T . M ania t i s . 1989 . Mo lecu la rC l o n i n g : A L a b o r a t o r y M a n u a l , 2 n d E d . C o l d S p r i n g H a r b o rL a b o r a t o r y Pr es s, C o l d S p r i n g H a r b o r , N Y .30 . Sak imu ra , K. , E . Kush iya , Y. Takahash i , and Y . Suzu k i .1987. T he s t ruc tu re and express ion o f the neuron -spec i f iceno lase gene . Gene (Amst.) . 6 0 : 1 0 3 - 1 1 3 .3 1 . A b r a h a m , C . R . , K . K a n e m u r u , a n d L . M u c k e . 1 9 93 . E x p r e s -s i o n o f c a t h e p s i n - G - l i k e a n d a l p h a - 1 a n t i c h y m o t r y p s i n l i k epro te ins in reac t ive as t rocy tes . Brain Res. 6 2 1 : 2 2 2 - 2 3 2 .32 . Al len , H . , J . F rase r , D. F lye r , S . Ca lv in , and R .A . F lave l l .1986. [3 -2 -m ic rog lob u l in is no t r equ i red fo r ce l l sur faceexpress ion o f the mu r ine c lass I h is tocom pat ib i l i ty an t igenH - 2 D b o r o f a t r u n c a t e d H - 2 D b . P roc. Na t l . A cad . Sc i . US A .8 2 : 7 4 4 7 - 7 4 5 1 .33 . Hogan , B . , F . Cons tan t in i , and E . Lacy . 1986 . Manipu la t ingt h e M o u s e G e n o m e , C o l d S p r i n g H a r b o r L a b o r a t o r y P re ss ,C o l d S p r i n g H a r b o r , N Y .3 4 . M u c k e , L . , M . B . A . O l d s t o n e , J . C . M o r r i s, a n d M . N e r e n -berg . 1991. R ap id ac t iva t ion o f a s t rocy te -spec i f ic express iono f G F A P - l a c Z t r a n s g e n e b y f o c a l i n j u r y . New B io l . 3 : 4 6 5 -474.3 5 . O l d s t o n e , M . B . A . , M . I . N e r e n b e r g , P J . S o u t h e r n , J . P r i c e ,and H. Lewick i . 1991 . Virus in fec t ion t r iggers insu l in depen-den t d iabe tes mel l i tu s in a transgen ic mode l : ro le o f an t i - se l f

(v i res ) immune response . Cell. 6 5 : 3 1 9 - 3 3 1 .3 6 . B a n k e r , G . , a n d K . G o s l i n . 1 9 91 . R a t h i p p o c a m p a l n e u r o n s

in low dens i ty cu l tu re . In C u l t u r i n g N e r v e C e l l s . G . B a n k e ra n d K . G o s l i n , ed i t o rs . T h e M I T P re ss , C a m b r i d g e , M A .2 5 1 - 2 8 1 .

37 . Pas ick , J .M .M . , K . Ka l ichar ran , and S . Da les . 1994 . Dis t r ibu -t i o n a n d t r a f f ic k i n g o f J H M c o r o n a v i m s s t r u ct u r a l p r o t e i n sa n d v i r i on s i n p r im a r y n e u r o n s a n d t h e O B L - 2 1 n e u r o n a l c e l ll i n e . J . Virol. 6 8 : 2 9 1 5 - 2 9 2 8 .

3 8 . M a c L e i s h , P . R . , C . J . B a m s t a b l e , a n d E . T o w n e s - A n d e r s o n .1 9 83 . U s e o f a m o n o c l o n a l a n t i b o d y a s a s u b s tr a t e f o r m a t u r eneurons in v i t ro . Proc. Nat l . Acad. Sci . US A. 8 0 : 7 0 1 4 - 7 0 1 8 .39 . O M al ley , M.B . , and P .R . MacLei~h . 1993 . Indu c t ion o f c lass

I m a j o r h i s t o c o m p a t i b i l i t y c o m p l e x a n t ig e n s o n a d u l t p r i m a t er e t i n al n e u r o n s . J . Neuroimmunol . 4 3 : 4 5 - 5 8 .4 0 . R a i l , G . F . , L . M u c k e , M . N e r e n b e r g , a n d M . B . A . O l d s t o n e .1994. A t ransgen ic mouse m ode l to assess the in te rac t ion o fc y t o t o x i c T l y m p h o c y t e s w i t h v i r a ll y i n f e c te d , c la ss I M H C -express ing as t rocy tes . J . Neuroimmunol . 5 2 : 4 1 - 5 0 .

41 . Olds tone , M.B .A. , P . B loun t , P . J . Sou thern , and P .W. Lamper t .1 9 86 . C y t o i m m u n o t h e r a p y f o r p e r s i s te n t v ir a l i n f e c t i o n r e -veals a un iqu e c lea rance pa t te rn f rom the cen t ra l ne rvous sys -t e m . Nature (Lond.). 3 2 1 : 2 3 9 - 2 4 3 .

4 2 . R o d r i g u e z , M . , M . J . B u c h m e i e r , M . B . A . O l d s t o n e , a n dP .W . L amper t . 1983. U l t ras t ruc tu ra l loca l iza t ion o f v i ra l an t i -gens in the cen t ra l ne rvous sys tem of mice pe rs i s ten t ly in -f e c te d w i t h L C M V . A m.J . P a tho l . 110 :95-100 .

43 . Fazaker ley , J .K. , P . J. Sou th ern , F .E . B loo m, a nd M.J . Buc h-meie r . 1991. H igh reso lu t ion in s i tu hybr id iza t io n to de te r -m i n e t h e c e l l u l a r d i s t r i b u t io n o f L C M V R N A i n t h e t is su e sof pe rs i s ten t ly in fec ted m ice : re leva nce to a renav i rus d iseaseand m echan isms o f v i ra l pe rs i s tence . J. Gen. Virol . 7 2 : 1 6 1 1 -1625.4 4 . T i s h o n , A . , M . E d d e l s t o n, J . C . d e l a T o r r e , a n d M . B . A . O l d -s tone . 1993 . Cy to tox ic T ly mph ocy tes c leanse v i ra l gene p ro -d u c t s f r o m i n d i v i d u a l l y in f e c t e d n e u r o n s a n d l y m p h o c y t e s i nm i c e p e r s i s t e n tl y i n f e c t e d w i t h L C M V . Virology. 1 9 7 : 4 6 3 -467.

4 5 . W a t t s , R . G . , J . L . W r i g h t , L .L . A t k i n s o n , a n d R . E . M e r c h a n t .1989. His top a tho log ica l and b loo d b ra in ba r r ie r changes inr at s i n d u c e d b y a n i n t r a c e r e b r a l i n j e c t i o n o f h u m a n r e c o m b i -n a n t i n t e r l e u k i n - 2 . Neurosurgery (Baltimore). 2 5 : 2 0 2 - 2 0 8 .

4 6 . C a m p b e l l , I . L . , C . R . A b r a h a m , E . M a s l ia h , P . K e m p e r , J . D .I n g li s , M . B . A . O l d s t o n e , a n d L . M u c k e . 1 9 93 . N e u r o l o g i cd isease induced in t ransgen ic mice by ce rebra l overexpres -s i on o f i n t e r l e u k i n - 6 . P roc. Na t l . A cad . Sc i . US A . 9 0 : 1 0 0 6 1 -10065.

47 . Gi l le s , P .N . , G . Fey , and F .V. Ch isa r i . 1992 . T um or nec ros isfac to r a lpha nega t ive ly regu la tes hepa t i t i s B v i rus gene expres -s i o n i n t r a n s g e ni c m i c e . J . Virol. 6 6 : 3 9 5 5 - 3 9 6 0 .48 . Gu i lho t , S . , L .G. Guido t t i , and F .V. Ch isa r i . 1993 . ln te r leu -k in -2 down regu la tes hepa t i t i s B v i rus gene express ion int ransgen ic mice by a pos t t ransc r ip t iona l mechan ism. J. Virol.6 7 : 7 4 4 4 - 7 4 4 9 .4 9 . G u i d o t t i , L . G . , K . A n d o , M . V . H o b b s , T . I s h i ka w a , L .R u n k e l , R . D . S c h r e i b e r, a n d F .V . C h i s a ri . 1 9 94 . C y t o t o x i cT lymphocy tes inh ib i t hepa t i t i s B v i rus gene express ion by an o n c y t o l y t i c m e c h a n i s m i n t r a n s g e n i c m i c e . Pro Nat l . Acad.Sc i . USA . 9 1 : 3 7 6 4 - 3 7 6 8 .

50 . Hseu h , F . , C . W alker , D. B lackb oum , and J . Levy . 1994 .S u p p r e s si o n o f H I V r e p l i c a t io n b y C D 8 + c e ll c l o n es d e r i v e df r o m H I V - i n f e c t e d a n d u n i n f e c t e d i n d i v i d u a ls . Cell. Immunol.159 :271-279 .

1211 Ra i l e t a l .


12/12

51. Rapo port, S.I. 1976. Blood-Brain Barrier in Physiology andMedicine. Raven Press, New York. 316 pp.

52. Pappius, H.M., and W. Feindel. 1976. Dynamic Aspects ofBrain Edema. Springer, Heidelberg and Berlin. 18-22.53. Miller, J.D., and J.H. Adams. 1992. The pathophysiology of

raised intracranial pressure. In Greenfield s Neuropathology.Fifth ed. J.H. Adams and L.W. Duchen, editors. OxfordUniversity Press, New York. 69-105.

54. Power, C., P.A. Kong, T.O. Crawford, S. Wesselingh, J.D.Glass, J.C. McArthur, and B.D. Trapp. 1993. Cerebral whitematter changes in AIDS dementia: alterations of the bloodbrain barrier. Ann Neurol 34:339-350.

55. Chaturvedi, U.C., R. Dhawan, M. Khanna, and A. Mathur.1991. Breakdown of the blood brain barrier during Denguevirus infection ofmice.J. Gen Virol 72:859-866.56. Mathur, A., N. Khanna, and U.C. Chaturvedi. 1992. Break-down of blood brain barrier by virus-in duced cytokines dur-ing Japanese encephalitis virus infec tion. Int d Exp Pathol

73:603--611.57. Anderson, I.H., O. Marker, and A.tk. Thomsen. 1991. Break-

down of blood brain barrier function in the murine LCMVinfection mediated by virus-specific CD8 T cells. ]. ~M,u-roimmunol 31:155-163.

58. So ilu-Ha nninen, M., J.p. Earlinna, V. Hukkanen , M. Roytta,A.A. Salmi, and R. Salonen. 1994. Semliki forest virus infectsmouse brain endothelial cells and causes blood-brai n barrierdamage.J. ViroI 68:6291-6298.

59. Sharief, M.K., M.A. Noori, M. Ciardi, A. Cirelli, and E.J.Thompson. 1993. Increased levels of circulating ICAM-1 inserum and CSF of patients with active mult iple sclerosis: cor-relation with TNF alpha and blood brain barrier damage, d.Neuroimmunol 43:15-22.

6{}. Adams, J.H., and L,W. Duch en, editors. 1992 . Greenfield sNeuropathology. Fifth ed. Oxford Unive rsity Press, NewYork. 1557 pp.

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