Immunological Communications
ISSN: 0090-0877 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/iimm19
Effect on Synthetic Capacity of Antibodies Directed Against Receptors of Allotype-Bearing Rabbit Cells Louise T. Adler To cite this article: Louise T. Adler (1976) Effect on Synthetic Capacity of Antibodies Directed Against Receptors of Allotype-Bearing Rabbit Cells, Immunological Communications, 5:4, 223-242, DOI: 10.3109/08820137609044278 To link to this article: http://dx.doi.org/10.3109/08820137609044278
Published online: 07 Jul 2009.
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Date: 17 April 2016, At: 22:29
IMMUNOLOGICAL COMMUNICATIONS, 5 ( 4 ) , 2 2 3 - 2 4 2
(1976)
EFFECT ON S Y N T H E T I C C A P A C I T Y OF A N T I B O D I E S D I R E C T E D A G A I N S T RECEPTORS O F ALLOTYPE-BEARIrdG RABBIT CELLS
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L o u i s e T. A d l e r D i v i s i o n o f Immunology S t . Jude C h i l d r e n ' s Research H o s p i t a l Memphis , Tennessee 381 01
R a b b i t i n i n i u n o g l o b u l i n m o l e c u l e s possess a n t i g e n i c a l l y d i s t i n c t a l l o t y p i c markers. I m m u n o g l o b u l i n m o l e c u l e s p o s s e s s i n g such m a r k e r s which are attached t o t h e surfaces o f lymphocyte precursors o f antibodyf o r m i n g c e l l s a r e b e l i e v e d t o s e r v e as a n t i g e n r e c e p t o r s . Interactions between a1 l o t y p i c d e t e r m i n a n t s and a n t i b o d i e s d i r e c t e d a g a i n s t them can p r o v i d e u s e f u l tnodels f o r t h e s t u d y o f i m m u n o r e g u l a t o r y phenomena. E x posure o f a f e t a l o r n e o n a t a l r a b b i t t o a n t i b o d i e s a g a i n s t one o f i t s immunoglobulin a l l o t y p e s r e s u l t s i n a p e r s i s t e n t d e f e c t i n expression o f t h a t t y p e ( a l l o t y p e s u p p r e s s i o n ) . Some e v i d e n c e i n d i c a t e s t h a t t h i s phenomenon i n v o l v e s s p e c i f i c a c t i v e s u p p r e s s i o n by l y m p h o i d c e l l s . By examining t h e e f f e c t s o f a n t i - a l l o t y p e a n t i b o d i e s on a l l o t y p e - b e a r i n q lymphocytes i n v i t r o , c e r t a i n s p e c i f i c e v e n t s s u r r o u n d i n p t h e p r o c e s s e s o f l y m p h o c y t e ontogeny, and t h e d i f f e r e n t i a t i o n and m a t u r a t i o n o f antibody-forming c e l l s a r e being analyzed. The o b s e r v a t i o n t h a t t h e r e a r e g e n e t i c a l l y d e t e r m i n e d a n t i g e n i c d i f f e r e n c e s among serum i m m u n o g l o b u l i n s o f i n d i v i d u a l r a b b i t s was f i r s t made by Oudin i n 1956 ( 1 ) .
S i n c e t h a t t i m e t h e i d e n t i t y and i n some cases
t h e l o c a t i o n o f a number o f such a n t i g e n i c d e t e r m i n a n t s on t h e imrnunoglob u l i n ( I g ) m o l e c u l e have been e s t a b l i s h e d ( 2 ) .
A t the present time
r a b b i t a l l o t y p e s have been i d e n t i f i e d on l i g h t c h a i n s o f t h e kappa and lambda c l a s s e s , as w e l l as on heavy c h a i n s o f gamma, mu, and a l p h a classes.
Antibodies against a given a l l o t y p e are g e n e r a l l y obtained by
i m m u n i z a t i o n o f r a b b i t s w h i c h do n o t s y n t h e s i z e I g molecules, w i t h t h a t determinant.
A l l o t y p e s encoded by t h e b l o c u s ( d e s i g n a t e d b 4 , b5, b6 and
223 Copyright 0 1976 hy Marcel Dekker, Inc All Rights Reserved. Neither this work nor any part may he reproduced or transmitted in any form or hy any means, electronic or mechanical. including photocopying. microfilming, and recording, or by any information storage and retrieval system, without permission i n writing from the publisher.
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b9 and l o c a t e d on kappa c h a i n s ) were among t h e e a r l i e s t d e s c r i b e d and have p r o b a b l y been used more e x t e n s i v e l y t h a n o t h e r a l l o t y p e s as m a r k e r s i n immunological s t u d i e s .
Most o f t h e work p r e s e n t e d i n t h i s r e v i e w d e a l s
w i t h b locus allotypes. A l l o t y p e Suppression i n V i v q
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A s t r i k i n g and f a r - r e a c h i n g e f f e c t o f a n t i b o d i e s a g a i n s t a l l o t y p i c markers was f i r s t n o t e d by Dray i n 1962 ( 3 ) .
His study involved the
i m m u n i z a t i o n o f f e m a l e r a b b i t s homozygous f o r t h e b 4 d e t e r m i n a n t a g a i n s t a n o t h e r o f t h e b l o c u s a l l e l i c p r o d u c t s , b5. mated w i t h homozygous b5 males.
These does were s u b s e q u e n t l y
The e n s u i n g o f f s p r i n g , g e n o t y p i c a l l y
b4b5, were, however, u n a b l e t o make I g o f t h e p a t e r n a l t y p e , presumably as a r e s u l t o f t h e i r exposure t o m a t e r n a l a n t i b o d i e s a g a i n s t t h i s gene product during i n t r a u t e r i n e l i f e .
No a d v e r s e e f f e c t s on t h e g e n e r a l w e l l -
b e i n g o f t h e progeny were n o t e d as a r e s u l t o f t h i s o n s l a u g h t by m a t e r n a l antibodies.
T o t a l I g l e v e l s were m a i n t a i n e d a t t h e normal l e v e l t h r o u g h
a compensatory o v e r p r o d u c t i o n o f t h e m a t e r n a l t y p e .
One o f t h e most
f a s c i n a t i n g a s p e c t s o f D r a y ' s o b s e r v a t i o n was t h a t t h i s c o n d i t i o n o f s p e c i f i c " a l l o t y p e s u p p r e s s i o n " p e r s i s t e d i n some degree f o r a t l e a s t a y e a r , even though m a t e r n a l a n t i b o d y was n o t d e t e c t a b l e i n t h e serum o f t h e young r a b b i t s a f t e r t h e age o f 8 weeks.
S u b s e q u e n t l y , i t was shown
t h a t t h e same e f f e c t c o u l d be b r o u g h t a b o u t by t h e i n j e c t i o n o f newborns w i t h a n t i b o d i e s a g a i n s t one o f t h e i r serum a l l o t y p e s ( 4 ) .
Suppression
i n v o l v i n g o t h e r a l l o t y p i c markers was soon d e m o n s t r a t e d ( 5 ) , and s u p p r e s s i o n o f homozygotes a l s o p r o v e d t o be p o s s i b l e when e x p e r i m e n t a l p r o c e d u r e s were devised t o circumvent t h e presence o f p a s s i v e l y d e r i v e d maternal I g i n t h e c i r c u l a t i o n o f homozygous neonates ( 6 - 8 ) .
The s u b j e c t o f a l l o t y p e s u p p r e s -
s i o n i n r a b b i t s has r e c e n t l y been t h o r o u g h l y r e v i e w e d b y Dray ( 9 ) and Mage (10- 1 2 ) .
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ANTIBODIES E F F E C T O N SYNTHETIC CAPACITY
I n V i t r o Models f o r A l l o t y p e SuppreJL& A t t e m p t s t o r e v e a l t h e n a t u r e o f t h e iiiechanism w h i c h [ m a i n t a i n s s u p p r e s s i o n have o c c u p i e d t h e c e n t e r o f i n t e r e s t i n a number o f l a b o r a t o r i e s f o r many y e a r s .
A t t h e h e a r t o f t h e m a t t e r , o f course, i s t h e q u e s t i o n o f
t h e n a t u r e o f t h e i n t e r a c t i o n between t h e a n t i - a l l o t y p i c a n t i b o d i e s w h i c h b r o u g h t a b o u t s u p p r e s s i o n and t h e i r t a r g e t i n t h e f e t a l o r n e o n a t a l r a b b i t .
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One k i n d o f approach t o t h i s p r o b l e n i has been t o examine t h e e f f e c t s o f such a n t i b o d i e s on r a b b i t l y m p h o i d c e l l s b e a r i n g a l l o t y p i c m a r k e r s i n various i n v i t r o settings.
These i n t e r a c t i o n s have a l s o been s t u d i e d i n
a more g e n e r a l c o n t e x t by t h o s e i n t e r e s t e d i n t h e o n t o g e n y o f a n t i b o d y f o r m i n g c e l l s o r i n fundamental p r i n c i p l e s o f immune r e g u l a t i o n r a t h e r t h a n i n t h e phenomenon o f a l l o t y p e s u p p r e s s i o n p e r s e .
This section w i l l
deal p r i m a r i l y w i t h t h e r e s u l t s o b t a i n e d by t h e s e groups o f i n v e s t i g a t o r s . S c h u f f l e r and Dray s e t o u t t o d e v e l o p an i n v i t r o model i n o r d e r t o a n a l y z e t h e mechanism o f a l l o t y p e s u p p r e s s i o n i n t h e i n t a c t a n i m a l ( 1 3 ) . They exposed c u l t u r e d s p l e e n c e l l s f r o m normal b4b5 r a b b i t s t o a n t i - b 4 o r a n t i - b 5 a n t i b o d y f o r t h e f i r s t 24 h r o f c u l t u r e , t h e n washed t h e c e l l s , and d e t e r m i n e d t h e c o n c e n t r a t i o n s o f b 4 and b5 I g f o r m e d by t h e c e l l s a f t e r a f u r t h e r 4 days o f c u l t u r e .
P r o d u c t i o n o f I g b e a r i n g each m a r k e r was
d e t e r m i n e d b y i n h i b i t i o n o f p r e c i p i t a t i o n i n an i n d i r e c t radioinimunoassay system.
S p e c i f i c a l l o t y p e s u p p r e s s i o n was o b s e r v e d , i n t h a t a n t i - b 4 i n h i -
b i t e d b4 p r o d u c t i o n w i t h n o e f f e c t on b5 I g l e v e l s , w h i l e t h e s o l e e f f e c t o f a n t i - b 5 i n t h e s e e x p e r i m e n t s was t o suppress p r o d u c t i o n o f b5 I g . analogous f a s h i o n a n t i b o d i e s a g a i n s t " a " l o c u s d e t e r m i n a n t s ,
In
l o c a t e d on
t h e Fd f r a g m e n t o f heavy c h a i n s were shown t o i n h i b i t s p e c i f i c a l l y p r o d u c t i o n o f the corresponding a l l o t y p e s o n l y (14). I n h i b i t i o n was a l s o o b s e r v e d when F ( a b ' ) 2 o r Fab f r a g m e n t s o f a n t i - b 4 IgG were t e s t e d a g a i n s t b4b5 s p l e e n c e l l s ( 1 5 ) .
Attempts t o induce a l l o t y p e
s u p p r e s s i o n i n v i v o u s i n g F ( a b ' ) 2 f r a g m e n t s o f a n t i - a l l o t y p e a n t i b o d y had
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p r e v i o u s l y been unsuccessful ( 1 6 ) , b u t t h i s can be a t t r i b u t e d t o t h e r a p i d e l i m i n a t i o n o f such fragments i n t h e i n t a c t animal ( 1 7 ) .
I t has
been observed r e p e a t e d l y t h a t a n t i - a l l o t y p e a n t i b o d i e s a r e n o t d i r e c t l y c y t o t o x i c f o r t a r g e t c e l l s marked w i t h t h e corresponding a l l o t y p e s (18-20). We have devised an i n v i t r o model f o r a l l o t y p e suppression s t u d i e s which d i f f e r s from t h a t o f S c h u f f l e r and Dray i n t h a t r e s u l t s a r e expressed
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i n terms o f a n t i b o d y responses by c u l t u r e d spleen c e l l s r a t h e r than t o t a l I g l e v e l s (21).
Spleen c e l l s o f unprimed animals a r e s t i m u l a t e d w i t h an
a n t i g e n o b t a i n e d by s o l u b i l i z a t i o n o f T2 bacteriophage, and t h e T2-neut r a l i z i n g a n t i b o d i e s formed can be b o t h i d e n t i f i e d and t i t r a t e d by means o f an a m p l i f i c a t i o n assay which uses s p e c i f i c a n t i - a l l o t y p e s e r a (22, 2 3 ) . We f e e l j u s t i f i e d i n u s i n g t h e a n t i - 2 response as a v a l i d i n d i c a t o r o f t h e t o t a l I g p r o d u c t i o n o f a g i v e n a l o t y p e , s i n c e we have c o n s i s t e n t l y observed t h a t t h e d i s t r i b u t i o n o f a1 otypes i n a n t i b o d i e s formed by c e l l s o f a heterozygous spleen donor r o u g h l y p a r a l l e l s t h e d i s t r i b u t i o n o f t h e a l l o t y p e s i n t h e serum o f t h a t animal ( 2 3 , 24).
This f i n d i n g r e f l e c t s the
probable m u l t i - d e t e r m i n a n t n a t u r e o f o u r a n t i g e n , a s u b j e c t discussed by Gel1 ( 2 5 ) , and c o n t r a s t s s t r i k i n g l y w i t h t h e pronounced tendency o f c e r t a i n antigens t o stimulate s e l e c t i v e l y c e l l s o f a given a l l o t y p e (26, 27). Using t h i s system we have confirmed t h e b a s i c f i n d i n g o f S c h u f f l e r and Dray r e g a r d i n g t h e s p e c i f i c i t y o f a l l o t y p e suppression i n v i t r o .
That i s ,
a n t i - b 5 serum prevented t h e f o r m a t i o n o f a n t i - T 2 w i t h t h e b5 marker by spleen c e l l s from b4b5 r a b b i t s , b u t d i d n o t suppress p r o d u c t i o n o f b4 anti-T2.
A n t i - b 4 e x e r t e d an e n t i r e l y analogous s p e c i f i c e f f e c t on b4
anti-T2 formation.
A d d i t i o n a l l y , an i n t r i g u i n g o b s e r v a t i o n was made w i t h
t h e c e l l s of a few b4b5 r a b b i t s which had u n u s u a l l y skewed ra.tios o f b4 t o b5 i n t h e i r serum.
As n o t e d e a r l i e r by Mage ( 2 8 ) , Chou e t a l . (29-31), and
o t h e r s , a n a t u r a l " p e c k i n g o r d e r " e x i s t s i n t h e r e l a t i v e serum concentrat i o n s of t h e two a l l e l i c types expressed by a heterozygous r a b b i t ; f o r
ANTIBODIES EFFECT ON SYNTHETIC CAPACITY example, b4 > b5 and b6 -, b9.
227
This suggests t h e occurrence o f a n a t u r a l l y
o c c u r r i n g r e g u l a t o r y phenomenon, f o r w h i c h a r t i f i c i a l l y i n d u c e d a1 l o t y p e s u p p r e s s i o n ( i n v i v o ) may s e r v e as an e x a g g e r a t e d model.
I n o u r hands,
when s p l e e n c e l l s o f r a b b i t s w i t h u n u s u a l l y h i g h serum r a t i o s o f b 4 t o b5 were t r e a t e d w i t h a n t i - b 4 serum, a h i g h l y s i g n i f i c a n t e l e v a t i o n o f t h e b5 a n t i - T 2 response o c c u r r e d ( 2 1 ) .
T h i s seemed t o be analogous t o t h e compensa-
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t o r y p r o d u c t i o n o f t h e a l t e r n a t e a l l o t y p e seen i n a l l o t y p e s u p p r e s s i o n ~
fi
v i v o , and c o n t r a s t s w i t h t h e absence o f a compensatory e f f e c t seen i n S c h u f f l e r and D r a y ' s e x p e r i m e n t s . Probable C e l l u l a r E v e n t s D u r i n g A l l o t y p e Suppression A l t h o u g h i t i s c l e a r f r o m t h e above d a t a t h a t
repression o f a l l o t y p e
s y n t h e s i s can be r e a d i l y i n d u c e d i n v i t r o , d e t a i l s o f t h e i n h i b i t i o n mechanism on a m o l e c u l a r and c e l l u l a r l e v e l a r e n o t w e l l u n d e r s t o o d .
A number o f
i n v e s t i g a t o r s have d e m o n s t r a t e d t h e p r e s e n c e o f a l l o t y p e - b e a r i n g m o l e c u l e s (presumably I g o r I g - l i k e r e c e p t o r s ) on t h e s u r f a c e o f r a b b i t l y m p h o i d c e l l s (32-34).
The i n i t i a t i n g e v e n t i n a l l o t y p e s u p p r e s s i o n c o u l d t h e n b e assumed
t o be t h e a t t a c h m e n t o f a n t i - a l l o t y - p e a n t i b o d y t o t h e a l l o t y p i c d e t e r m i n a n t o f a membrane-bound I g r e c e p t o r .
The sequence o f e v e n t s f o l l o w i n g b i n d i n g
o f l a b e l e d a n t i - I g t o mouse s p l e e n c e l l s has been s t u d i e d i n some d e t a i l (35-37).
U n i f o r m b i n d i n g w i t h some p a t c h y a r e a s o f l a b e l i n g was soon
f o l l o w e d by t h e a g g r e g a t i o n o f most o r a l l o f t h e l a b e l a t one p o l e o f t h e c e l l ("capping").
Cap f o r m a t i o n , w h i c h o c c u r r e d a t 37°C b u t n o t a t O°C,
was r a p i d l y f o l l o w e d b y p i n o c y t o s i s o f t h e a n t i b o d y - I g complex.
This
reaction l e f t the c e l l temporarily depleted o f surface receptors. p r o b a b l y r e l a t e d phenomenon i s t h a t o f " a n t i g e n i c m o d u l a t i o n . "
A
As o b s e r v e d
by O l d and h i s c o l l e a g u e s (38, 3 9 ) , t h i s c o n s i s t s o f t h e d i s a p p e a r a n c e o f thymus-leukemia ( T L ) a n t i g e n f r o m t h e s u r f a c e s o f mouse l y m p h o i d c e l l s a f t e r exposure t o a n t i - T L a n t i s e r u m .
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L i n t h i c u m and S e l l (40, 4 1 ) used an i n d i r e c t i m n i u n o f e r r i t i n l a b e l i n g t e c h n i q u e t o s t u d y t h e i n t e r a c t on between l y m p h o i d c e l l s f r o m homozygous b4 r a b b i t s w i t h a n t i - b 4 .
Per p h e r a l b l o o d lymphocytes bound
t h e a n t i b o d y i n s p a r s e p a t c h e s a t 0”C, and warminq t h e c e l l s t o 37°C caused r a p i d e n d o c y t o s i s w i t h o u t cap f o r m a t i o n .
The a u t h o r s suggested t h a t t h e
absence o f cap f o r i n a t i o n m i g h t be a t t r i b u t a b l e t o t h e o b s e r v e d s p a r s e
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d i s t r i b u t i o n o f a l l o t y p e - b e a r i n g r e c e p t o r s on t h e c e l l membrane o f t h e s e c e l l s , a c o n d i t i o n w h i c h w o u l d make c r o s s - l i n k a g e i n t o one l a r g e a g g r e g a t e unlikely.
A l a r g e percentage o f s p l e n i c lymphocytes e x h i b i t e d surface
l a b e l i n g p r o p e r t i e s s i m i l a r t o those o f p e r i p h e r a l blood c e l l s . The d i s a p p e a r a n c e o f s u r f a c e r e c e p t o r s a f t e r t h e i r u n i o n w i t h a n t i - I g r e a g e n t s , f o l l o w e d by t h e g e n e r a t i o n o f new r e c e p t o r s , a c c o r d s w e l l w i t h t h e o b s e r v e d i n v i t r o e f f e c t s o f a n t i - a l l o t y p e serum t r e a t m e n t .
It i s
c l e a r f r o m s t u d y i n g t h e e f f e c t s o f a n t i - a l l o t y p e a n t i b o d i e s on c e l l s t h a t t h e s i n g l e event o f b i n d i n g o f a n t i b o d i e s t o a l l o t y p e - b e a r i n g I g molecules ( r e c e p t o r s ) does n o t r e s u l t i n p e r m a n e n t l y b l o c k i n g t h o s e a c t i v i t i e s o f t h e c e l l i n which receptors a r e i n v o l v e d .
Prolonged exposure t o a n t i - a l l o t y p e
a n t i b o d i e s seems t o be r e q u i r e d i n o r d e r t o cause c o m p l e t e shutdown o f s p e c i f i c I g o r a n t i b o d y s y n t h e s i s (13, 2 1 ) .
I t seems most l i k e l y f r o m t h e
m i c r o s c o p i c o b s e r v a t i o n s c i t e d t h a t a f t e r b i n d i n g o f a n t i b o d y and aggreg a t i o n i n t o p a t c h e s o r caps, f o l l o w e d by e n d o c y t o s i s o r s h e d d i n g o f r e c e p t o r s , prompt r e g e n e r a t i o n o f t h e r e c e p t o r s l e a d s t o a r e p e t i t i o n o f t h i s e n t i r e process.
I n t h e c o n t i n u e d presence o f a n t i b o d i e s , an e v e n t u a l
exhaustion o f t h e regenerative processes o f t h e c e l l c o u l d very w e l l occur, b u t such a breakdown may r e q u i r e a s e r i e s o f membrane r e g e n e r a t i o n c y c l e s . S c h u f f l e r and Dray ( 1 3 ) f o u n d i t n e c e s s a r y t o c u l t u r e b4b5 s p l e e n c e l l s w i t h a n t i - b 4 o r a n t i - b 5 a n t i b o d y f o r 24 h o u r s i n o r d e r t o a c h i e v e maximum i n h i b i t i o n o f I g synthesis.
A 4 h o u r i n c u b a t i o n p e r i o d under t h e i r s t a n d a r d
c o n d i t i o n s f o r i n v i t r o a l l o t y p e s u p p r e s s i o n was o n l y p a r t i a l l y e f f e c t i v e .
229
ANTIBODIES EFFECT ON SYNTHETIC CAPACITY
We have s t u d i e d t h e k i n e t i c s o f i n d u c t i o n o f a l l o t y p e s u p p r e s s i o n i n v i t r o and have n o t e d t h a t exposure of homozygous b4 s p l e e n c e l l s t o a p o t e n t a n t i - b 4 serum f o r as l i t t l e as one h o u r may c o m p l e t e l y i n h i b i t t h e c a p a c i t y o f t h e s e c e l l s t o form n e u t r a l i z i n g a n t i b o d i e s when t h e y a r e s u b s e q u e n t l y s t i m u l a t e d w i t h s o l u b i l i z e d T2 phage ( 2 1 ) .
W i t h s m a l l e r doses o r l e s s
p o t e n t a n t i s e r a , l o n g e r exposure t i m e s a r e r e q u i r e d t o e f f e c t c o m p l e t e
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suppress ion. The u l t i m a t e f a t e o f c e l l s s u b j e c t e d t o an i r r e v e r s i b l e t r e a t m e n t w i t h s p e c i f i c a n t i - a l l o t y p e serum i s n o t known.
As m e n t i o n e d e a r l i e r i n
t h i s a r t i c l e , no d i r e c t e v i d e n c e f o r a c y t o t o x i c e f f e c t by a n t i - a l l o t y p e serum has been n o t e d ( 1 8 - 2 0 ) .
S i m i l a r o b s e r , v a t i o n s were made w i t h r e g a r d
t o a n t i s e r a d i r e c t e d a g a i n s t mouse a l l o a n t i g e n s by P i e r c e e t a l . (42), i n t h a t p r o l o n g e d exposure t o such s e r a i n t e r f e r e d w i t h t h e responses o f mouse s p l e e n c e l l s t o sheep e r y t h r o c y t e s i n v i t r o , w i t h o u t any demons t r a b l e c y t o t o x i c a c t i o n on t h e c e l l s .
However, i t i s p o s s i b l e t h a t c e l l s
w h i c h do n o t appear t o be dead when t h e c r i t e r i o n o f dye e x c l u s i o n i s used c o u l d be m e t a b o l i c a l l y i n e r t due t o t h e i n t e r f e r e n c e o f a n t i - a l l o t y p e a n t i bodies w i t h s y n t h e t i c processes.
A p o s s i b l e p a r a l l e l may e x i s t
i n the
i n d u c t i o n o f t o l e r a n c e on a c e l l u l a r l e v e l and t h e g e n e r a t i o n o f t h e socalled "tolerant cell ."
Considerable evidence i n d i c a t e s t h a t exposure o f
antigen-reactive c e l l s t o a high concentration o f antigen leads t o a c o n d i t i o n o f i m m u n o l o g i c a l p a r a l y s i s w h i c h i,;c h a r a c t e r i z e d by t h e i n a b i l i t y o f t h e s e c e l l s t o r e g e n e r a t e r e c e p t o r s and t h e r e t e n t i o n o f a n t i g e n o n t h e membrane ( 4 3 - 4 6 ) . E n t i r e l y d i f f e r e n t c r i t e r i a have been used by S e l l and h i s c o l l e a g u e s t o assess t h e e f f e c t s o f a n t i - a l l o t y p i c a n t i b o d i e s on a l l o t y p e - b e a r i n g r a b b i t lymphoid c e l l s (47-52).
They have r e p e a t e d l y o b s e r v e d s t i m u l a t i o n
o f DNA s y n t h e s i s and changes a s s o c i a t e d w i t h t y p i c a l " b l a s t " c e l l morphology as a r e s u l t o f such i n t e r a c t i o n .
Anti-allotype stimulation o f rabbit peri-
2 30
ADLER
p h e r a l b l o o d lymphocytes was f o u n d t o be c o m p l e t e l y r e v e r s i b l e up t o 36 o r 42 h o u r s o f i n i t i a t i o n by t h e a d d i t i o n o f serum c o n t a i n i n g t h e
a l l o t y p i c s p e c i f i c i t y t o w a r d w h i c h t h e a n t i s e r u m was d i r e c t e d ( 5 3 ) . These o b s e r v a t i o n s may v e r y w e l l have b e a r i n g on t h e mechanism l e a d i n g t o i n h i b i t i o n o f I g s y n t h e s i s b y a n t i - a l l o t y p e serum.
As p o s t u l a t e d by
Gel1 ( 2 5 ) and o t h e r s , s t i m u l a t i o n o f a l l o t y p e - p r o d u c i n g c e l l s b y a n t i -
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a l l o t y p e serum may cause i n a c t i v a t i o n o f such c e l l s t h r o u g h an i r r e v e r s i b l e and a b o r t i v e b l a s t t r a n s f o r m a t i o n p r o c e s s . P o s s i b l e D i f f e r e n c e s Between A d u l t and Immature Lymphocytes
A l l o f t h e i n v i t r o s t u d i e s d e s c r i b e d t h u s f a r have d e a l t w i t h t h e e f f e c t s o f a n t i - a l l o t y p e serum on l y m p h o i d c e l l s o f a d u l t r a b b i t s .
However,
t h e i n v i v o i n d u c t i o n o f a l l o t y p e s u p p r e s s i o n succeeds o n l y d u r i n g a c r i t i c a l p e r i o d o f e a r l y l i f e (54, 55).
I f one o f t h e g o a l s o f t h e i n v i t r o s t u d i e s
u n d e r d i s c u s s i o n i s t o shed some l i g h t o n t h e mechanism o f a l l o t y p e s u p p r e s s i o n i n t h e i n t a c t a n i m a l , p o s s i b l e d i f f e r e n c e s between a d u l t and f e t a l o r n e o n a t a l c e l l s need t o be c o n s i d e r e d .
I t i s w e l l known t h a t a
newborn r a b b i t possesses no d e t e c t a b l e I g o f i t s own.
Active I g synthesis
b e g i n s o n t h e f i r s t day o f l i f e ( 5 6 ) , b u t f o r t h e f i r s t week o r two t h i s i s o n l y a s m a l l f r a c t i o n o f t h e t o t a l c o n c e n t r a t i o n , most o f w h i c h has been obtained passively from the maternal c i r c u l a t i o n .
Maternal I g i s r a p i d l y
c a t a b o l i z e d d u r i n g t h e f i r s t month, and a r o u n d t h a t t i m e a l l o t y p e s encoded by t h e young r a b b i t ' s genotype a r e e a s i l y d e t e c t a b l e b y i m m u n o l o g i c a l methods ( 3 , 57).
C e l l s marked w i t h t h e s e a l l o t y p e s a r e p r e s e n t i n l o w
numbers d u r i n g t h e n e o n a t a l p e r i o d and g r a d u a l l y r e a c h a d u l t l e v e l s d u r i n g t h e f i r s t 3 t o 4 months o f l i f e ( 5 7 , 5 8 ) .
The p r o l i f e r a t i v e responses t o
a n t i - a l l o t y p e serum and t o m i t o g e n s i s p r e s e n t i n l y m p h o i d c e l l s o f newborns b u t do n o t a t t a i n a d u l t l e v e l s u n t i l a b o u t t h e age o f one month ( 5 8 ) .
The
i n j e c t i o n o f a n t i - a l l o t y p e serum i n t o newborn h e t e r o z y g o t e s causes a p r o m p t
ANTIBODIES EFFECT ON SYNTHETIC CAPACITY
231
d i s a p p e a r a n c e o f c e l l s marked w i t h t h e c o r r e s p o n d i n g a t l o t y p e f r o m t h e l y m p h o i d o r g a n s (57, 5 9 ) .
F u r t h e r m o r e , t h e lymphocytes f r o m such a l l o -
t y p i c a l l y suppressed r a b b i t s e x h i b i t a decreased a b i l i t y t o underqo b l a s t t r a n s f o r m a t i o n when exposed t o a n t i s e r u r r i a g a i n s t t h e suppressed type (50).
I t i s t e m p t i n g t o s p e c u l a t e t h a t a l l o t y p e s u p p r e s s i o n may
be i n d u c i b l e i n v i v o o n l y v e r y e a r l y i n l i t e because o f q u a n t i t a t i v e o r
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q u a l i t a t i v e d i f f e r e n c e s i n t h e s u r f a c e p r o p e r t i e s o f a d u l t as opposed t o immature c e l l s .
Recent e v i d e n c e t o s u g g e s t such d i f f e r e n c e s comes f r o m
an o b s e r v a t i o n t h a t s p l e e n c e l l s o f i n f a n t m i c e r e g e n e r a t e s u r f a c e I g p o o r l y , i f a t a l l , a f t e r c l e a r a n c e by a n t i - I g ( 6 0 ) . The E f f e c t s o f H e t e r o l o g o u s A n t i - A l l o t y p i c A n t i b o d i e s A n t i - a l l o t y p e s e r a p r e p a r e d i n s p e c i e s o t h e r t h a n t h e r a b b i t have p r o v e d t o be a l m o s t c o m p l e t e l y w i t h o u t e f f e c t i n i n d u c i n g a l l o t y p e s u p p r e s s i o n i n newborn r a b b i t s .
Lowe ( 1 8 ) r e a c h e d t h i s c o n c l u s i o n u s i n g a sheep
a n t i - b 6 l i g h t c h a i n serum, s u i t a b l y a b s o r b e d t o remove o t h e specificities.
anti-rabbit
T h i s serum r e a d i l y i n d u c e d b l a s t t r a n s f o r m a i o n i n b6 lym-
p h o c y t e s i n v i t r o , whereas r a b b i t a n t i s e r u m s p e c i f i c f o r b6 l i g h t c h a i n s i n d u c e d b o t h b6 s u p p r e s s i o n i n v i v o and b l a s t t r a n s f o r m a t i o n i n v i t r o . Goldman and Mage ( 5 4 ) a c h i e v e d o n l y m a r g i n a l a l l o t y p e s u p p r e s s i o n by a d m i n i s t e r i n g m u l t i p l e i n j e c t i o n s o f g o a t a n t i - b 5 and a n t i - b 4 s e r a t o newborn b4b5 r a b b i t s .
Such i n t e n s i v e t r e a t m e n t caused o n l y s l i g h t tempor-
a r y delays i n attainment o f the a d u l t l e v e l o f t h e a f f e c t e d a l l o t y p e . P u r i f i e d g o a t a n t i - a l l o t y p e a n t i b o d i e s were s i m i l a r l y i n e f f e c t i v e . I n t e r e s t i n g l y , when p u r i f i e d r a b b i t a n t i b o d y f r a c t i o n s were t e s t e d f o r t h e i r a b i l i t y t o i n d u c e a l l o t y p e s u p p r e s s i o n i n neonates, a t o t a l o f 45 mg o f p r e c i p i t a b l e a n t i - b 5 g i v e n o v e r a p e r i o d o f s e v e r a l days was needed t o b r i n g a b o u t c o m p l e t e b5 s u p p r e s s i o n .
In contrast, the equivalent o f less
t h a n 5 mg o f a n t i b o d y a c h i e v e d t h e same r e s u l t when u n f r a c t i o n a t e d serum
232
ADLER
was used.
The a u t h o r s o f t h i s p a p e r a t t r i b u t e d t h e i r r e s u l t s t o t h e l o w
s t a b i l i t y o f p u r i f i e d a n t i b o d i e s and t o t h e more r a p i d d e g r a d a t i o n o f heterologous than
homologous p r o t e i n s i n v i v o .
This conclusion i s i n
a c c o r d w i t h Lowe's o b s e r v a t i o n ( 1 8 ) t h a t h e t e r o l o g o u s a n t i - a 1 l o t y p e serum does i n t e r a c t i n a d e m o n s t r a b l e way w i t h a l l o t y p e - b e a r i n g l y m p h o c y t e s j r ~
e, if not
i n vivo.
R e c e n t l y , Shek and D u b i s k i ( 6 1 ) have p r e s e n t e d
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e v i d e n c e f o r a c o m p e t i t i v e b i n d i n g e f f e c t on t a r g e t l y m p h o c y t e s between h e t e r o l o g o u s a n t i b o d i e s o r F ( a b ' ) * f r a g m e n t s o f r a b b i t a n t i b o d i e s and i n t a c t r a b b i t a n t i b o d i e s capable o f i n d u c i n q a l l o t y p e suppression.
This
s t r o n g l y i m p l i e s t h a t t h e c a p a c i t y t o i n d u c e s u p p r e s s i o n i n v i v o does n o t r e s i d e s o l e l y i n a b i l i t y t o b i n d t o t a r g e t lymphocytes b u t t h a t an i n t a c t Fc p o r t i o n o f r a b b i t a n t i b o d y i s p r o b a b l y r e q u i r e d . E f f e c t s o f A n t i - A l l o t y p e Serum on C e l l s From A l l o t y p e - S u p p r e s s e d R a b b i t s
A m a j o r p a r t o f o u r approach t o t h e p r o b l e m o f a l l o t y p e s u p p r e s s i o n has c e n t e r e d on t h e r e a c t i v i t i e s o f l y m p h o i d c e l l s f r o m p r e n a t a l l y s u p p r e s s e d r a b b i t s i n t h e c u l t u r e system d e s c r i b e d i n an e a r l i e r s e c t i o n .
Ever s i n c e
a l l o t y p e s u p p r e s s i o n was f i r s t d e s c r i b e d ( 3 ) , w o r k e r s i n t h i s a r e a o f r e s e a r c h have been s t r u c k by t h e l o n g - l a s t i n g n a t u r e o f t h e s u p p r e s s i v e effect i n relation t o the rapid elimination o f the inducing anti-allotype a g e n t , an o b s e r v a t i o n w h i c h made i t d i f f i c u l t t o a c c e p t c o n t i n u e d t a r g e t c e l l e l i m i n a t i o n as t h e c a u s a t i v e mechanism.
The f i n d i n g o f o t h e r models
f o r a l l o t y p e s u p p r e s s i o n i n c e r t a i n mouse s t r a i n c o m b i n a t i o n s (62, 6 3 ) , and i n p a r t i c u l a r t h e d e m o n s t r a t i o n t h a t s p e c i f i c s u p p r e s s i o n can b e i n d u c e d i n normal s y n g e n e i c r e c i p i e n t s by t h e t r a n s f e r o f v i a b l e c e l l s f r o m suppressed m i c e ( 6 4 , 6 5 ) p r o v i d e d a f i r m base f o r t h e e x c i t i n g c o n c e p t o f an a c t i v e s u p p r e s s i o n mechanism m e d i a t e d by s u p p r e s s o r c e l l s .
The
s c a r c i t y o f s u f f i c i e n t i n b r e d r a b b i t s has d i s c o u r a g e d a n a l o g o u s d i r e c t t e s t s f o r suppressor c e l l s .
However, e x p e r i m e n t a l o b s e r v a t i o n s made i n
ANTIBODIES EFFECT ON SYNTHETIC CAPACITY
233
two l a b o r a t o r i e s i n d e p e n d e n t l y p r o v i d e d j u s t i f i c a t i o n f o r t h e i d e a o f an a c t i v e s u p p r r s s i o n inechanism i n r a b b i t s as w e l l . was t h e f i n d i n g by H a r r i s o n
The f i r s t o f t h e s e
( 6 6 ) , showing t h a t d u r i n g spontaneous
r e c o v e r y f r o m b5 s u p p r e s s i o n , l y m p h o c y t e s marked w i t h membrane b5 a p p r o a c h normal nunibers a t t h e same t i m e t h a t serum I g w i t h a l l o t y p e b5 i s s t i l l a t a very low l e v e l .
This observation l e d these i n v e s t i g a t o r s t o p o s t u l a t e
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t h e e x i s t e n c e o f a f u n c t i o n a l b l o c k i n a c t i v a t i o n o r m a t u r a t i o n o f b5b e a r i n g B lymphocytes i n b5-suppressed r a b b i t s w h i c h r e s u l t s i n d e f i c i e n t s e c r e t i o n o f b5.
A s i m i l a r c o n c l u s i o n was r e a c h e d b y A d l e r and Fishman
( 6 7 ) , who n o t e d t h a t t h e p o o l o f i n t r a c e l l u l a r I g i n lymph node c e l l s o f b4-suppressed b4b5 r a b b i t s c o n t a i n e d a much h i g h e r p r o p o r t i o n o f t h e suppressed a l l o t y p e t h a n was i n d i c a t e d by t h e serum r a t i o o f b4 t o b5 concentrations.
Further,
t h e i r studies indicated that the block i n
c h r o n i c s u p p r e s s i o n i s l o c a t e d somewhere beyond s y n t h e s i s o f messenger RNA r e l e v a n t t o t h e suppressed a l l o t y p e .
O t h e r e x p e r i m e n t s done i n o u r
l a b o r a t o r y , t o be d e s c r i b e d below, have p r o v i d e d more d i r e c t e v i d e n c e f o r an a c t i v e s u p p r e s s i o n mechanism i n s p l e e n c e l l s o f a l l o t y p e - s u p p r e s s e d rabbits. We have r o u t i n e l y i n d u c e d a l l o t y p e s u p p r e s s i o n i n r a b b i t s w h i c h were h e t e r o z y g o u s a t t h e b (kappa c h a i n ) l o c u s b y i m m u n i z i n g homozygous f e m a l e s a g a i n s t t h e b l o c u s a l l o t y p e o f t h e i r p r o s p e c t i v e homozygous mates.
Most
o f t h e p r o g e n y o b t a i n e d b y t h i s method have been c o m p l e t e l y s u p p r e s s e d i n terms o f t h e p a t e r n a l t y p e when f i r s t t e s t e d a t w e a n i n g age ( 8 weeks), u s i n g a t e s t w h i c h d e t e c t s as l i t t l e as 1 pg o f a g i v e n a l l o t y p i c s p e c i f i c i t y (23). T a b l e I l i s t s t h e a l l o t y p i c c o m b i n a t i o n s we have u t i l i z e d t o b r e e d the rabbits f o r our studies.
Data summarized i n T a b l e I 1 show t h a t , i n
each i n s t a n c e , s p l e e n c e l l s t a k e n f r o m an a l l o t y p e - s u p p r e s s e d r a b b i t formed n e u t r a l i z i n g a n t i b o d i e s w h i c h were marked w i t h t h e m a t e r n a l (non-
234
TABLE I Breeding Schemes f o r A l l o t y p e Suppression
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Dam -
Sire
Progeny
b5, a n t i - b 4
b4
( b 4 ) b5
b6, a n t i - b 4
b4
(b4)b6
b6, a n t i - b 5
b5
( b 5 ) b6
b5, a n t i - b 6
b6
b5(b6)
b4, a n t i - b 5
b5
b4(b5)
b4, a n t i - b 6
b6
b4( b6)
The suppressed t y p e o f t h e o f f s p r i n g i s shown i n parentheses.
suppressed) a l l o t y p e o n l y , when t h e y were s t i m u l a t e d w i t h t h e s o l u b l e T2 a n t i g e n (S-T2) i n c u l t u r e .
C e l l s o f normal h e t e r o z y g o t e s formed a n t i -
bodies o f both a l l o t y p i c s p e c i f i c i t i e s encoded by t h e i r genotype.
I n another s e r i e s o f experiments spleen c e l l s o f (b4)b5 (b4-suppressed b4b5) r a b b i t s were
exposed t o
anti-b5
serum
(made i n b4 r a b b i t s )
d u r i n g t h e e n t i r e c u l t u r e p e r i o d o f 4 t o 5 days.
As observed p r e v i o u s l y
when normal b4b5 spleen were c u l t u r e d i n t h e presence o f a : i t i - b 5 , a marked i n h i b i t i o n o f b5 a n t i - T 2 p r o d u c t i o n ( 2 1 ) .
t h e r e was
Surprisingly, i n h i b i t i o n
o f t h e non-suppressed a l l o t y p e ( b 5 ) was accompanied by t h e appearance o f h i g h l y s i g n i f i c a n t t i t e r s c o n t r i b u t e d by b4 a n t i - T 2 ( 2 3 ) .
The d i s t r i b u t i o n
o f a l l o t y p i c s p e c i f i c i t i e s between b4 and b5 a n t i - T 2 a c t i v i t i e s o b t a i n e d under these c o n d i t i o n s o f t e n resembled t h a t formed by normal b4b5 spleen c e l l s (Figure 1).
These r e s u l t s p r o v i d e d d i r e c t evidence f o r t h e
e x i s t e n c e o f an a c t i v e suppression mechanism which c o n t i n u e d t o e x e r t i t s e f f e c t i n v i t r o on c e l l s p o t e n t i a l l y capable o f making t h e suppressed a l l o t y p e , and which c o u l d seemingly be overcome by suppressing t h e a c t i v i t i e s of c e l l s a s s o c i a t e d w i t h p r o d u c t i o n o f t h e non-suppressed I g t y p e .
ANTIBODIES EFFECT ON SYNTHETIC CAPACITY
235
TABLE I 1 A l l o t y p e D i s t r i b u t i o n o f T2 N e u t r a l i z i n g A n t i b o d i e s Made i n Spleen C e l l - C u l t u r e s f r o m Normal and A j j o t y p e - S u p p r e s s e d R a b b i t s A l l o t y p e ( s ) o f Spleen Donor
Grolrp
R e c i p r o c a l o f 302 N e u t r a l i z a t i o n T i t e r * b4 a n t i - T 2 b5 a n t i - T Z b6 a n t i - T 2 ~-
b4b4
64
ISSN: 0090-0877 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/iimm19
Effect on Synthetic Capacity of Antibodies Directed Against Receptors of Allotype-Bearing Rabbit Cells Louise T. Adler To cite this article: Louise T. Adler (1976) Effect on Synthetic Capacity of Antibodies Directed Against Receptors of Allotype-Bearing Rabbit Cells, Immunological Communications, 5:4, 223-242, DOI: 10.3109/08820137609044278 To link to this article: http://dx.doi.org/10.3109/08820137609044278
Published online: 07 Jul 2009.
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Date: 17 April 2016, At: 22:29
IMMUNOLOGICAL COMMUNICATIONS, 5 ( 4 ) , 2 2 3 - 2 4 2
(1976)
EFFECT ON S Y N T H E T I C C A P A C I T Y OF A N T I B O D I E S D I R E C T E D A G A I N S T RECEPTORS O F ALLOTYPE-BEARIrdG RABBIT CELLS
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L o u i s e T. A d l e r D i v i s i o n o f Immunology S t . Jude C h i l d r e n ' s Research H o s p i t a l Memphis , Tennessee 381 01
R a b b i t i n i n i u n o g l o b u l i n m o l e c u l e s possess a n t i g e n i c a l l y d i s t i n c t a l l o t y p i c markers. I m m u n o g l o b u l i n m o l e c u l e s p o s s e s s i n g such m a r k e r s which are attached t o t h e surfaces o f lymphocyte precursors o f antibodyf o r m i n g c e l l s a r e b e l i e v e d t o s e r v e as a n t i g e n r e c e p t o r s . Interactions between a1 l o t y p i c d e t e r m i n a n t s and a n t i b o d i e s d i r e c t e d a g a i n s t them can p r o v i d e u s e f u l tnodels f o r t h e s t u d y o f i m m u n o r e g u l a t o r y phenomena. E x posure o f a f e t a l o r n e o n a t a l r a b b i t t o a n t i b o d i e s a g a i n s t one o f i t s immunoglobulin a l l o t y p e s r e s u l t s i n a p e r s i s t e n t d e f e c t i n expression o f t h a t t y p e ( a l l o t y p e s u p p r e s s i o n ) . Some e v i d e n c e i n d i c a t e s t h a t t h i s phenomenon i n v o l v e s s p e c i f i c a c t i v e s u p p r e s s i o n by l y m p h o i d c e l l s . By examining t h e e f f e c t s o f a n t i - a l l o t y p e a n t i b o d i e s on a l l o t y p e - b e a r i n q lymphocytes i n v i t r o , c e r t a i n s p e c i f i c e v e n t s s u r r o u n d i n p t h e p r o c e s s e s o f l y m p h o c y t e ontogeny, and t h e d i f f e r e n t i a t i o n and m a t u r a t i o n o f antibody-forming c e l l s a r e being analyzed. The o b s e r v a t i o n t h a t t h e r e a r e g e n e t i c a l l y d e t e r m i n e d a n t i g e n i c d i f f e r e n c e s among serum i m m u n o g l o b u l i n s o f i n d i v i d u a l r a b b i t s was f i r s t made by Oudin i n 1956 ( 1 ) .
S i n c e t h a t t i m e t h e i d e n t i t y and i n some cases
t h e l o c a t i o n o f a number o f such a n t i g e n i c d e t e r m i n a n t s on t h e imrnunoglob u l i n ( I g ) m o l e c u l e have been e s t a b l i s h e d ( 2 ) .
A t the present time
r a b b i t a l l o t y p e s have been i d e n t i f i e d on l i g h t c h a i n s o f t h e kappa and lambda c l a s s e s , as w e l l as on heavy c h a i n s o f gamma, mu, and a l p h a classes.
Antibodies against a given a l l o t y p e are g e n e r a l l y obtained by
i m m u n i z a t i o n o f r a b b i t s w h i c h do n o t s y n t h e s i z e I g molecules, w i t h t h a t determinant.
A l l o t y p e s encoded by t h e b l o c u s ( d e s i g n a t e d b 4 , b5, b6 and
223 Copyright 0 1976 hy Marcel Dekker, Inc All Rights Reserved. Neither this work nor any part may he reproduced or transmitted in any form or hy any means, electronic or mechanical. including photocopying. microfilming, and recording, or by any information storage and retrieval system, without permission i n writing from the publisher.
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b9 and l o c a t e d on kappa c h a i n s ) were among t h e e a r l i e s t d e s c r i b e d and have p r o b a b l y been used more e x t e n s i v e l y t h a n o t h e r a l l o t y p e s as m a r k e r s i n immunological s t u d i e s .
Most o f t h e work p r e s e n t e d i n t h i s r e v i e w d e a l s
w i t h b locus allotypes. A l l o t y p e Suppression i n V i v q
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A s t r i k i n g and f a r - r e a c h i n g e f f e c t o f a n t i b o d i e s a g a i n s t a l l o t y p i c markers was f i r s t n o t e d by Dray i n 1962 ( 3 ) .
His study involved the
i m m u n i z a t i o n o f f e m a l e r a b b i t s homozygous f o r t h e b 4 d e t e r m i n a n t a g a i n s t a n o t h e r o f t h e b l o c u s a l l e l i c p r o d u c t s , b5. mated w i t h homozygous b5 males.
These does were s u b s e q u e n t l y
The e n s u i n g o f f s p r i n g , g e n o t y p i c a l l y
b4b5, were, however, u n a b l e t o make I g o f t h e p a t e r n a l t y p e , presumably as a r e s u l t o f t h e i r exposure t o m a t e r n a l a n t i b o d i e s a g a i n s t t h i s gene product during i n t r a u t e r i n e l i f e .
No a d v e r s e e f f e c t s on t h e g e n e r a l w e l l -
b e i n g o f t h e progeny were n o t e d as a r e s u l t o f t h i s o n s l a u g h t by m a t e r n a l antibodies.
T o t a l I g l e v e l s were m a i n t a i n e d a t t h e normal l e v e l t h r o u g h
a compensatory o v e r p r o d u c t i o n o f t h e m a t e r n a l t y p e .
One o f t h e most
f a s c i n a t i n g a s p e c t s o f D r a y ' s o b s e r v a t i o n was t h a t t h i s c o n d i t i o n o f s p e c i f i c " a l l o t y p e s u p p r e s s i o n " p e r s i s t e d i n some degree f o r a t l e a s t a y e a r , even though m a t e r n a l a n t i b o d y was n o t d e t e c t a b l e i n t h e serum o f t h e young r a b b i t s a f t e r t h e age o f 8 weeks.
S u b s e q u e n t l y , i t was shown
t h a t t h e same e f f e c t c o u l d be b r o u g h t a b o u t by t h e i n j e c t i o n o f newborns w i t h a n t i b o d i e s a g a i n s t one o f t h e i r serum a l l o t y p e s ( 4 ) .
Suppression
i n v o l v i n g o t h e r a l l o t y p i c markers was soon d e m o n s t r a t e d ( 5 ) , and s u p p r e s s i o n o f homozygotes a l s o p r o v e d t o be p o s s i b l e when e x p e r i m e n t a l p r o c e d u r e s were devised t o circumvent t h e presence o f p a s s i v e l y d e r i v e d maternal I g i n t h e c i r c u l a t i o n o f homozygous neonates ( 6 - 8 ) .
The s u b j e c t o f a l l o t y p e s u p p r e s -
s i o n i n r a b b i t s has r e c e n t l y been t h o r o u g h l y r e v i e w e d b y Dray ( 9 ) and Mage (10- 1 2 ) .
225
ANTIBODIES E F F E C T O N SYNTHETIC CAPACITY
I n V i t r o Models f o r A l l o t y p e SuppreJL& A t t e m p t s t o r e v e a l t h e n a t u r e o f t h e iiiechanism w h i c h [ m a i n t a i n s s u p p r e s s i o n have o c c u p i e d t h e c e n t e r o f i n t e r e s t i n a number o f l a b o r a t o r i e s f o r many y e a r s .
A t t h e h e a r t o f t h e m a t t e r , o f course, i s t h e q u e s t i o n o f
t h e n a t u r e o f t h e i n t e r a c t i o n between t h e a n t i - a l l o t y p i c a n t i b o d i e s w h i c h b r o u g h t a b o u t s u p p r e s s i o n and t h e i r t a r g e t i n t h e f e t a l o r n e o n a t a l r a b b i t .
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One k i n d o f approach t o t h i s p r o b l e n i has been t o examine t h e e f f e c t s o f such a n t i b o d i e s on r a b b i t l y m p h o i d c e l l s b e a r i n g a l l o t y p i c m a r k e r s i n various i n v i t r o settings.
These i n t e r a c t i o n s have a l s o been s t u d i e d i n
a more g e n e r a l c o n t e x t by t h o s e i n t e r e s t e d i n t h e o n t o g e n y o f a n t i b o d y f o r m i n g c e l l s o r i n fundamental p r i n c i p l e s o f immune r e g u l a t i o n r a t h e r t h a n i n t h e phenomenon o f a l l o t y p e s u p p r e s s i o n p e r s e .
This section w i l l
deal p r i m a r i l y w i t h t h e r e s u l t s o b t a i n e d by t h e s e groups o f i n v e s t i g a t o r s . S c h u f f l e r and Dray s e t o u t t o d e v e l o p an i n v i t r o model i n o r d e r t o a n a l y z e t h e mechanism o f a l l o t y p e s u p p r e s s i o n i n t h e i n t a c t a n i m a l ( 1 3 ) . They exposed c u l t u r e d s p l e e n c e l l s f r o m normal b4b5 r a b b i t s t o a n t i - b 4 o r a n t i - b 5 a n t i b o d y f o r t h e f i r s t 24 h r o f c u l t u r e , t h e n washed t h e c e l l s , and d e t e r m i n e d t h e c o n c e n t r a t i o n s o f b 4 and b5 I g f o r m e d by t h e c e l l s a f t e r a f u r t h e r 4 days o f c u l t u r e .
P r o d u c t i o n o f I g b e a r i n g each m a r k e r was
d e t e r m i n e d b y i n h i b i t i o n o f p r e c i p i t a t i o n i n an i n d i r e c t radioinimunoassay system.
S p e c i f i c a l l o t y p e s u p p r e s s i o n was o b s e r v e d , i n t h a t a n t i - b 4 i n h i -
b i t e d b4 p r o d u c t i o n w i t h n o e f f e c t on b5 I g l e v e l s , w h i l e t h e s o l e e f f e c t o f a n t i - b 5 i n t h e s e e x p e r i m e n t s was t o suppress p r o d u c t i o n o f b5 I g . analogous f a s h i o n a n t i b o d i e s a g a i n s t " a " l o c u s d e t e r m i n a n t s ,
In
l o c a t e d on
t h e Fd f r a g m e n t o f heavy c h a i n s were shown t o i n h i b i t s p e c i f i c a l l y p r o d u c t i o n o f the corresponding a l l o t y p e s o n l y (14). I n h i b i t i o n was a l s o o b s e r v e d when F ( a b ' ) 2 o r Fab f r a g m e n t s o f a n t i - b 4 IgG were t e s t e d a g a i n s t b4b5 s p l e e n c e l l s ( 1 5 ) .
Attempts t o induce a l l o t y p e
s u p p r e s s i o n i n v i v o u s i n g F ( a b ' ) 2 f r a g m e n t s o f a n t i - a l l o t y p e a n t i b o d y had
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p r e v i o u s l y been unsuccessful ( 1 6 ) , b u t t h i s can be a t t r i b u t e d t o t h e r a p i d e l i m i n a t i o n o f such fragments i n t h e i n t a c t animal ( 1 7 ) .
I t has
been observed r e p e a t e d l y t h a t a n t i - a l l o t y p e a n t i b o d i e s a r e n o t d i r e c t l y c y t o t o x i c f o r t a r g e t c e l l s marked w i t h t h e corresponding a l l o t y p e s (18-20). We have devised an i n v i t r o model f o r a l l o t y p e suppression s t u d i e s which d i f f e r s from t h a t o f S c h u f f l e r and Dray i n t h a t r e s u l t s a r e expressed
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i n terms o f a n t i b o d y responses by c u l t u r e d spleen c e l l s r a t h e r than t o t a l I g l e v e l s (21).
Spleen c e l l s o f unprimed animals a r e s t i m u l a t e d w i t h an
a n t i g e n o b t a i n e d by s o l u b i l i z a t i o n o f T2 bacteriophage, and t h e T2-neut r a l i z i n g a n t i b o d i e s formed can be b o t h i d e n t i f i e d and t i t r a t e d by means o f an a m p l i f i c a t i o n assay which uses s p e c i f i c a n t i - a l l o t y p e s e r a (22, 2 3 ) . We f e e l j u s t i f i e d i n u s i n g t h e a n t i - 2 response as a v a l i d i n d i c a t o r o f t h e t o t a l I g p r o d u c t i o n o f a g i v e n a l o t y p e , s i n c e we have c o n s i s t e n t l y observed t h a t t h e d i s t r i b u t i o n o f a1 otypes i n a n t i b o d i e s formed by c e l l s o f a heterozygous spleen donor r o u g h l y p a r a l l e l s t h e d i s t r i b u t i o n o f t h e a l l o t y p e s i n t h e serum o f t h a t animal ( 2 3 , 24).
This f i n d i n g r e f l e c t s the
probable m u l t i - d e t e r m i n a n t n a t u r e o f o u r a n t i g e n , a s u b j e c t discussed by Gel1 ( 2 5 ) , and c o n t r a s t s s t r i k i n g l y w i t h t h e pronounced tendency o f c e r t a i n antigens t o stimulate s e l e c t i v e l y c e l l s o f a given a l l o t y p e (26, 27). Using t h i s system we have confirmed t h e b a s i c f i n d i n g o f S c h u f f l e r and Dray r e g a r d i n g t h e s p e c i f i c i t y o f a l l o t y p e suppression i n v i t r o .
That i s ,
a n t i - b 5 serum prevented t h e f o r m a t i o n o f a n t i - T 2 w i t h t h e b5 marker by spleen c e l l s from b4b5 r a b b i t s , b u t d i d n o t suppress p r o d u c t i o n o f b4 anti-T2.
A n t i - b 4 e x e r t e d an e n t i r e l y analogous s p e c i f i c e f f e c t on b4
anti-T2 formation.
A d d i t i o n a l l y , an i n t r i g u i n g o b s e r v a t i o n was made w i t h
t h e c e l l s of a few b4b5 r a b b i t s which had u n u s u a l l y skewed ra.tios o f b4 t o b5 i n t h e i r serum.
As n o t e d e a r l i e r by Mage ( 2 8 ) , Chou e t a l . (29-31), and
o t h e r s , a n a t u r a l " p e c k i n g o r d e r " e x i s t s i n t h e r e l a t i v e serum concentrat i o n s of t h e two a l l e l i c types expressed by a heterozygous r a b b i t ; f o r
ANTIBODIES EFFECT ON SYNTHETIC CAPACITY example, b4 > b5 and b6 -, b9.
227
This suggests t h e occurrence o f a n a t u r a l l y
o c c u r r i n g r e g u l a t o r y phenomenon, f o r w h i c h a r t i f i c i a l l y i n d u c e d a1 l o t y p e s u p p r e s s i o n ( i n v i v o ) may s e r v e as an e x a g g e r a t e d model.
I n o u r hands,
when s p l e e n c e l l s o f r a b b i t s w i t h u n u s u a l l y h i g h serum r a t i o s o f b 4 t o b5 were t r e a t e d w i t h a n t i - b 4 serum, a h i g h l y s i g n i f i c a n t e l e v a t i o n o f t h e b5 a n t i - T 2 response o c c u r r e d ( 2 1 ) .
T h i s seemed t o be analogous t o t h e compensa-
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t o r y p r o d u c t i o n o f t h e a l t e r n a t e a l l o t y p e seen i n a l l o t y p e s u p p r e s s i o n ~
fi
v i v o , and c o n t r a s t s w i t h t h e absence o f a compensatory e f f e c t seen i n S c h u f f l e r and D r a y ' s e x p e r i m e n t s . Probable C e l l u l a r E v e n t s D u r i n g A l l o t y p e Suppression A l t h o u g h i t i s c l e a r f r o m t h e above d a t a t h a t
repression o f a l l o t y p e
s y n t h e s i s can be r e a d i l y i n d u c e d i n v i t r o , d e t a i l s o f t h e i n h i b i t i o n mechanism on a m o l e c u l a r and c e l l u l a r l e v e l a r e n o t w e l l u n d e r s t o o d .
A number o f
i n v e s t i g a t o r s have d e m o n s t r a t e d t h e p r e s e n c e o f a l l o t y p e - b e a r i n g m o l e c u l e s (presumably I g o r I g - l i k e r e c e p t o r s ) on t h e s u r f a c e o f r a b b i t l y m p h o i d c e l l s (32-34).
The i n i t i a t i n g e v e n t i n a l l o t y p e s u p p r e s s i o n c o u l d t h e n b e assumed
t o be t h e a t t a c h m e n t o f a n t i - a l l o t y - p e a n t i b o d y t o t h e a l l o t y p i c d e t e r m i n a n t o f a membrane-bound I g r e c e p t o r .
The sequence o f e v e n t s f o l l o w i n g b i n d i n g
o f l a b e l e d a n t i - I g t o mouse s p l e e n c e l l s has been s t u d i e d i n some d e t a i l (35-37).
U n i f o r m b i n d i n g w i t h some p a t c h y a r e a s o f l a b e l i n g was soon
f o l l o w e d by t h e a g g r e g a t i o n o f most o r a l l o f t h e l a b e l a t one p o l e o f t h e c e l l ("capping").
Cap f o r m a t i o n , w h i c h o c c u r r e d a t 37°C b u t n o t a t O°C,
was r a p i d l y f o l l o w e d b y p i n o c y t o s i s o f t h e a n t i b o d y - I g complex.
This
reaction l e f t the c e l l temporarily depleted o f surface receptors. p r o b a b l y r e l a t e d phenomenon i s t h a t o f " a n t i g e n i c m o d u l a t i o n . "
A
As o b s e r v e d
by O l d and h i s c o l l e a g u e s (38, 3 9 ) , t h i s c o n s i s t s o f t h e d i s a p p e a r a n c e o f thymus-leukemia ( T L ) a n t i g e n f r o m t h e s u r f a c e s o f mouse l y m p h o i d c e l l s a f t e r exposure t o a n t i - T L a n t i s e r u m .
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L i n t h i c u m and S e l l (40, 4 1 ) used an i n d i r e c t i m n i u n o f e r r i t i n l a b e l i n g t e c h n i q u e t o s t u d y t h e i n t e r a c t on between l y m p h o i d c e l l s f r o m homozygous b4 r a b b i t s w i t h a n t i - b 4 .
Per p h e r a l b l o o d lymphocytes bound
t h e a n t i b o d y i n s p a r s e p a t c h e s a t 0”C, and warminq t h e c e l l s t o 37°C caused r a p i d e n d o c y t o s i s w i t h o u t cap f o r m a t i o n .
The a u t h o r s suggested t h a t t h e
absence o f cap f o r i n a t i o n m i g h t be a t t r i b u t a b l e t o t h e o b s e r v e d s p a r s e
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d i s t r i b u t i o n o f a l l o t y p e - b e a r i n g r e c e p t o r s on t h e c e l l membrane o f t h e s e c e l l s , a c o n d i t i o n w h i c h w o u l d make c r o s s - l i n k a g e i n t o one l a r g e a g g r e g a t e unlikely.
A l a r g e percentage o f s p l e n i c lymphocytes e x h i b i t e d surface
l a b e l i n g p r o p e r t i e s s i m i l a r t o those o f p e r i p h e r a l blood c e l l s . The d i s a p p e a r a n c e o f s u r f a c e r e c e p t o r s a f t e r t h e i r u n i o n w i t h a n t i - I g r e a g e n t s , f o l l o w e d by t h e g e n e r a t i o n o f new r e c e p t o r s , a c c o r d s w e l l w i t h t h e o b s e r v e d i n v i t r o e f f e c t s o f a n t i - a l l o t y p e serum t r e a t m e n t .
It i s
c l e a r f r o m s t u d y i n g t h e e f f e c t s o f a n t i - a l l o t y p e a n t i b o d i e s on c e l l s t h a t t h e s i n g l e event o f b i n d i n g o f a n t i b o d i e s t o a l l o t y p e - b e a r i n g I g molecules ( r e c e p t o r s ) does n o t r e s u l t i n p e r m a n e n t l y b l o c k i n g t h o s e a c t i v i t i e s o f t h e c e l l i n which receptors a r e i n v o l v e d .
Prolonged exposure t o a n t i - a l l o t y p e
a n t i b o d i e s seems t o be r e q u i r e d i n o r d e r t o cause c o m p l e t e shutdown o f s p e c i f i c I g o r a n t i b o d y s y n t h e s i s (13, 2 1 ) .
I t seems most l i k e l y f r o m t h e
m i c r o s c o p i c o b s e r v a t i o n s c i t e d t h a t a f t e r b i n d i n g o f a n t i b o d y and aggreg a t i o n i n t o p a t c h e s o r caps, f o l l o w e d by e n d o c y t o s i s o r s h e d d i n g o f r e c e p t o r s , prompt r e g e n e r a t i o n o f t h e r e c e p t o r s l e a d s t o a r e p e t i t i o n o f t h i s e n t i r e process.
I n t h e c o n t i n u e d presence o f a n t i b o d i e s , an e v e n t u a l
exhaustion o f t h e regenerative processes o f t h e c e l l c o u l d very w e l l occur, b u t such a breakdown may r e q u i r e a s e r i e s o f membrane r e g e n e r a t i o n c y c l e s . S c h u f f l e r and Dray ( 1 3 ) f o u n d i t n e c e s s a r y t o c u l t u r e b4b5 s p l e e n c e l l s w i t h a n t i - b 4 o r a n t i - b 5 a n t i b o d y f o r 24 h o u r s i n o r d e r t o a c h i e v e maximum i n h i b i t i o n o f I g synthesis.
A 4 h o u r i n c u b a t i o n p e r i o d under t h e i r s t a n d a r d
c o n d i t i o n s f o r i n v i t r o a l l o t y p e s u p p r e s s i o n was o n l y p a r t i a l l y e f f e c t i v e .
229
ANTIBODIES EFFECT ON SYNTHETIC CAPACITY
We have s t u d i e d t h e k i n e t i c s o f i n d u c t i o n o f a l l o t y p e s u p p r e s s i o n i n v i t r o and have n o t e d t h a t exposure of homozygous b4 s p l e e n c e l l s t o a p o t e n t a n t i - b 4 serum f o r as l i t t l e as one h o u r may c o m p l e t e l y i n h i b i t t h e c a p a c i t y o f t h e s e c e l l s t o form n e u t r a l i z i n g a n t i b o d i e s when t h e y a r e s u b s e q u e n t l y s t i m u l a t e d w i t h s o l u b i l i z e d T2 phage ( 2 1 ) .
W i t h s m a l l e r doses o r l e s s
p o t e n t a n t i s e r a , l o n g e r exposure t i m e s a r e r e q u i r e d t o e f f e c t c o m p l e t e
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suppress ion. The u l t i m a t e f a t e o f c e l l s s u b j e c t e d t o an i r r e v e r s i b l e t r e a t m e n t w i t h s p e c i f i c a n t i - a l l o t y p e serum i s n o t known.
As m e n t i o n e d e a r l i e r i n
t h i s a r t i c l e , no d i r e c t e v i d e n c e f o r a c y t o t o x i c e f f e c t by a n t i - a l l o t y p e serum has been n o t e d ( 1 8 - 2 0 ) .
S i m i l a r o b s e r , v a t i o n s were made w i t h r e g a r d
t o a n t i s e r a d i r e c t e d a g a i n s t mouse a l l o a n t i g e n s by P i e r c e e t a l . (42), i n t h a t p r o l o n g e d exposure t o such s e r a i n t e r f e r e d w i t h t h e responses o f mouse s p l e e n c e l l s t o sheep e r y t h r o c y t e s i n v i t r o , w i t h o u t any demons t r a b l e c y t o t o x i c a c t i o n on t h e c e l l s .
However, i t i s p o s s i b l e t h a t c e l l s
w h i c h do n o t appear t o be dead when t h e c r i t e r i o n o f dye e x c l u s i o n i s used c o u l d be m e t a b o l i c a l l y i n e r t due t o t h e i n t e r f e r e n c e o f a n t i - a l l o t y p e a n t i bodies w i t h s y n t h e t i c processes.
A p o s s i b l e p a r a l l e l may e x i s t
i n the
i n d u c t i o n o f t o l e r a n c e on a c e l l u l a r l e v e l and t h e g e n e r a t i o n o f t h e socalled "tolerant cell ."
Considerable evidence i n d i c a t e s t h a t exposure o f
antigen-reactive c e l l s t o a high concentration o f antigen leads t o a c o n d i t i o n o f i m m u n o l o g i c a l p a r a l y s i s w h i c h i,;c h a r a c t e r i z e d by t h e i n a b i l i t y o f t h e s e c e l l s t o r e g e n e r a t e r e c e p t o r s and t h e r e t e n t i o n o f a n t i g e n o n t h e membrane ( 4 3 - 4 6 ) . E n t i r e l y d i f f e r e n t c r i t e r i a have been used by S e l l and h i s c o l l e a g u e s t o assess t h e e f f e c t s o f a n t i - a l l o t y p i c a n t i b o d i e s on a l l o t y p e - b e a r i n g r a b b i t lymphoid c e l l s (47-52).
They have r e p e a t e d l y o b s e r v e d s t i m u l a t i o n
o f DNA s y n t h e s i s and changes a s s o c i a t e d w i t h t y p i c a l " b l a s t " c e l l morphology as a r e s u l t o f such i n t e r a c t i o n .
Anti-allotype stimulation o f rabbit peri-
2 30
ADLER
p h e r a l b l o o d lymphocytes was f o u n d t o be c o m p l e t e l y r e v e r s i b l e up t o 36 o r 42 h o u r s o f i n i t i a t i o n by t h e a d d i t i o n o f serum c o n t a i n i n g t h e
a l l o t y p i c s p e c i f i c i t y t o w a r d w h i c h t h e a n t i s e r u m was d i r e c t e d ( 5 3 ) . These o b s e r v a t i o n s may v e r y w e l l have b e a r i n g on t h e mechanism l e a d i n g t o i n h i b i t i o n o f I g s y n t h e s i s b y a n t i - a l l o t y p e serum.
As p o s t u l a t e d by
Gel1 ( 2 5 ) and o t h e r s , s t i m u l a t i o n o f a l l o t y p e - p r o d u c i n g c e l l s b y a n t i -
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a l l o t y p e serum may cause i n a c t i v a t i o n o f such c e l l s t h r o u g h an i r r e v e r s i b l e and a b o r t i v e b l a s t t r a n s f o r m a t i o n p r o c e s s . P o s s i b l e D i f f e r e n c e s Between A d u l t and Immature Lymphocytes
A l l o f t h e i n v i t r o s t u d i e s d e s c r i b e d t h u s f a r have d e a l t w i t h t h e e f f e c t s o f a n t i - a l l o t y p e serum on l y m p h o i d c e l l s o f a d u l t r a b b i t s .
However,
t h e i n v i v o i n d u c t i o n o f a l l o t y p e s u p p r e s s i o n succeeds o n l y d u r i n g a c r i t i c a l p e r i o d o f e a r l y l i f e (54, 55).
I f one o f t h e g o a l s o f t h e i n v i t r o s t u d i e s
u n d e r d i s c u s s i o n i s t o shed some l i g h t o n t h e mechanism o f a l l o t y p e s u p p r e s s i o n i n t h e i n t a c t a n i m a l , p o s s i b l e d i f f e r e n c e s between a d u l t and f e t a l o r n e o n a t a l c e l l s need t o be c o n s i d e r e d .
I t i s w e l l known t h a t a
newborn r a b b i t possesses no d e t e c t a b l e I g o f i t s own.
Active I g synthesis
b e g i n s o n t h e f i r s t day o f l i f e ( 5 6 ) , b u t f o r t h e f i r s t week o r two t h i s i s o n l y a s m a l l f r a c t i o n o f t h e t o t a l c o n c e n t r a t i o n , most o f w h i c h has been obtained passively from the maternal c i r c u l a t i o n .
Maternal I g i s r a p i d l y
c a t a b o l i z e d d u r i n g t h e f i r s t month, and a r o u n d t h a t t i m e a l l o t y p e s encoded by t h e young r a b b i t ' s genotype a r e e a s i l y d e t e c t a b l e b y i m m u n o l o g i c a l methods ( 3 , 57).
C e l l s marked w i t h t h e s e a l l o t y p e s a r e p r e s e n t i n l o w
numbers d u r i n g t h e n e o n a t a l p e r i o d and g r a d u a l l y r e a c h a d u l t l e v e l s d u r i n g t h e f i r s t 3 t o 4 months o f l i f e ( 5 7 , 5 8 ) .
The p r o l i f e r a t i v e responses t o
a n t i - a l l o t y p e serum and t o m i t o g e n s i s p r e s e n t i n l y m p h o i d c e l l s o f newborns b u t do n o t a t t a i n a d u l t l e v e l s u n t i l a b o u t t h e age o f one month ( 5 8 ) .
The
i n j e c t i o n o f a n t i - a l l o t y p e serum i n t o newborn h e t e r o z y g o t e s causes a p r o m p t
ANTIBODIES EFFECT ON SYNTHETIC CAPACITY
231
d i s a p p e a r a n c e o f c e l l s marked w i t h t h e c o r r e s p o n d i n g a t l o t y p e f r o m t h e l y m p h o i d o r g a n s (57, 5 9 ) .
F u r t h e r m o r e , t h e lymphocytes f r o m such a l l o -
t y p i c a l l y suppressed r a b b i t s e x h i b i t a decreased a b i l i t y t o underqo b l a s t t r a n s f o r m a t i o n when exposed t o a n t i s e r u r r i a g a i n s t t h e suppressed type (50).
I t i s t e m p t i n g t o s p e c u l a t e t h a t a l l o t y p e s u p p r e s s i o n may
be i n d u c i b l e i n v i v o o n l y v e r y e a r l y i n l i t e because o f q u a n t i t a t i v e o r
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q u a l i t a t i v e d i f f e r e n c e s i n t h e s u r f a c e p r o p e r t i e s o f a d u l t as opposed t o immature c e l l s .
Recent e v i d e n c e t o s u g g e s t such d i f f e r e n c e s comes f r o m
an o b s e r v a t i o n t h a t s p l e e n c e l l s o f i n f a n t m i c e r e g e n e r a t e s u r f a c e I g p o o r l y , i f a t a l l , a f t e r c l e a r a n c e by a n t i - I g ( 6 0 ) . The E f f e c t s o f H e t e r o l o g o u s A n t i - A l l o t y p i c A n t i b o d i e s A n t i - a l l o t y p e s e r a p r e p a r e d i n s p e c i e s o t h e r t h a n t h e r a b b i t have p r o v e d t o be a l m o s t c o m p l e t e l y w i t h o u t e f f e c t i n i n d u c i n g a l l o t y p e s u p p r e s s i o n i n newborn r a b b i t s .
Lowe ( 1 8 ) r e a c h e d t h i s c o n c l u s i o n u s i n g a sheep
a n t i - b 6 l i g h t c h a i n serum, s u i t a b l y a b s o r b e d t o remove o t h e specificities.
anti-rabbit
T h i s serum r e a d i l y i n d u c e d b l a s t t r a n s f o r m a i o n i n b6 lym-
p h o c y t e s i n v i t r o , whereas r a b b i t a n t i s e r u m s p e c i f i c f o r b6 l i g h t c h a i n s i n d u c e d b o t h b6 s u p p r e s s i o n i n v i v o and b l a s t t r a n s f o r m a t i o n i n v i t r o . Goldman and Mage ( 5 4 ) a c h i e v e d o n l y m a r g i n a l a l l o t y p e s u p p r e s s i o n by a d m i n i s t e r i n g m u l t i p l e i n j e c t i o n s o f g o a t a n t i - b 5 and a n t i - b 4 s e r a t o newborn b4b5 r a b b i t s .
Such i n t e n s i v e t r e a t m e n t caused o n l y s l i g h t tempor-
a r y delays i n attainment o f the a d u l t l e v e l o f t h e a f f e c t e d a l l o t y p e . P u r i f i e d g o a t a n t i - a l l o t y p e a n t i b o d i e s were s i m i l a r l y i n e f f e c t i v e . I n t e r e s t i n g l y , when p u r i f i e d r a b b i t a n t i b o d y f r a c t i o n s were t e s t e d f o r t h e i r a b i l i t y t o i n d u c e a l l o t y p e s u p p r e s s i o n i n neonates, a t o t a l o f 45 mg o f p r e c i p i t a b l e a n t i - b 5 g i v e n o v e r a p e r i o d o f s e v e r a l days was needed t o b r i n g a b o u t c o m p l e t e b5 s u p p r e s s i o n .
In contrast, the equivalent o f less
t h a n 5 mg o f a n t i b o d y a c h i e v e d t h e same r e s u l t when u n f r a c t i o n a t e d serum
232
ADLER
was used.
The a u t h o r s o f t h i s p a p e r a t t r i b u t e d t h e i r r e s u l t s t o t h e l o w
s t a b i l i t y o f p u r i f i e d a n t i b o d i e s and t o t h e more r a p i d d e g r a d a t i o n o f heterologous than
homologous p r o t e i n s i n v i v o .
This conclusion i s i n
a c c o r d w i t h Lowe's o b s e r v a t i o n ( 1 8 ) t h a t h e t e r o l o g o u s a n t i - a 1 l o t y p e serum does i n t e r a c t i n a d e m o n s t r a b l e way w i t h a l l o t y p e - b e a r i n g l y m p h o c y t e s j r ~
e, if not
i n vivo.
R e c e n t l y , Shek and D u b i s k i ( 6 1 ) have p r e s e n t e d
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e v i d e n c e f o r a c o m p e t i t i v e b i n d i n g e f f e c t on t a r g e t l y m p h o c y t e s between h e t e r o l o g o u s a n t i b o d i e s o r F ( a b ' ) * f r a g m e n t s o f r a b b i t a n t i b o d i e s and i n t a c t r a b b i t a n t i b o d i e s capable o f i n d u c i n q a l l o t y p e suppression.
This
s t r o n g l y i m p l i e s t h a t t h e c a p a c i t y t o i n d u c e s u p p r e s s i o n i n v i v o does n o t r e s i d e s o l e l y i n a b i l i t y t o b i n d t o t a r g e t lymphocytes b u t t h a t an i n t a c t Fc p o r t i o n o f r a b b i t a n t i b o d y i s p r o b a b l y r e q u i r e d . E f f e c t s o f A n t i - A l l o t y p e Serum on C e l l s From A l l o t y p e - S u p p r e s s e d R a b b i t s
A m a j o r p a r t o f o u r approach t o t h e p r o b l e m o f a l l o t y p e s u p p r e s s i o n has c e n t e r e d on t h e r e a c t i v i t i e s o f l y m p h o i d c e l l s f r o m p r e n a t a l l y s u p p r e s s e d r a b b i t s i n t h e c u l t u r e system d e s c r i b e d i n an e a r l i e r s e c t i o n .
Ever s i n c e
a l l o t y p e s u p p r e s s i o n was f i r s t d e s c r i b e d ( 3 ) , w o r k e r s i n t h i s a r e a o f r e s e a r c h have been s t r u c k by t h e l o n g - l a s t i n g n a t u r e o f t h e s u p p r e s s i v e effect i n relation t o the rapid elimination o f the inducing anti-allotype a g e n t , an o b s e r v a t i o n w h i c h made i t d i f f i c u l t t o a c c e p t c o n t i n u e d t a r g e t c e l l e l i m i n a t i o n as t h e c a u s a t i v e mechanism.
The f i n d i n g o f o t h e r models
f o r a l l o t y p e s u p p r e s s i o n i n c e r t a i n mouse s t r a i n c o m b i n a t i o n s (62, 6 3 ) , and i n p a r t i c u l a r t h e d e m o n s t r a t i o n t h a t s p e c i f i c s u p p r e s s i o n can b e i n d u c e d i n normal s y n g e n e i c r e c i p i e n t s by t h e t r a n s f e r o f v i a b l e c e l l s f r o m suppressed m i c e ( 6 4 , 6 5 ) p r o v i d e d a f i r m base f o r t h e e x c i t i n g c o n c e p t o f an a c t i v e s u p p r e s s i o n mechanism m e d i a t e d by s u p p r e s s o r c e l l s .
The
s c a r c i t y o f s u f f i c i e n t i n b r e d r a b b i t s has d i s c o u r a g e d a n a l o g o u s d i r e c t t e s t s f o r suppressor c e l l s .
However, e x p e r i m e n t a l o b s e r v a t i o n s made i n
ANTIBODIES EFFECT ON SYNTHETIC CAPACITY
233
two l a b o r a t o r i e s i n d e p e n d e n t l y p r o v i d e d j u s t i f i c a t i o n f o r t h e i d e a o f an a c t i v e s u p p r r s s i o n inechanism i n r a b b i t s as w e l l . was t h e f i n d i n g by H a r r i s o n
The f i r s t o f t h e s e
( 6 6 ) , showing t h a t d u r i n g spontaneous
r e c o v e r y f r o m b5 s u p p r e s s i o n , l y m p h o c y t e s marked w i t h membrane b5 a p p r o a c h normal nunibers a t t h e same t i m e t h a t serum I g w i t h a l l o t y p e b5 i s s t i l l a t a very low l e v e l .
This observation l e d these i n v e s t i g a t o r s t o p o s t u l a t e
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t h e e x i s t e n c e o f a f u n c t i o n a l b l o c k i n a c t i v a t i o n o r m a t u r a t i o n o f b5b e a r i n g B lymphocytes i n b5-suppressed r a b b i t s w h i c h r e s u l t s i n d e f i c i e n t s e c r e t i o n o f b5.
A s i m i l a r c o n c l u s i o n was r e a c h e d b y A d l e r and Fishman
( 6 7 ) , who n o t e d t h a t t h e p o o l o f i n t r a c e l l u l a r I g i n lymph node c e l l s o f b4-suppressed b4b5 r a b b i t s c o n t a i n e d a much h i g h e r p r o p o r t i o n o f t h e suppressed a l l o t y p e t h a n was i n d i c a t e d by t h e serum r a t i o o f b4 t o b5 concentrations.
Further,
t h e i r studies indicated that the block i n
c h r o n i c s u p p r e s s i o n i s l o c a t e d somewhere beyond s y n t h e s i s o f messenger RNA r e l e v a n t t o t h e suppressed a l l o t y p e .
O t h e r e x p e r i m e n t s done i n o u r
l a b o r a t o r y , t o be d e s c r i b e d below, have p r o v i d e d more d i r e c t e v i d e n c e f o r an a c t i v e s u p p r e s s i o n mechanism i n s p l e e n c e l l s o f a l l o t y p e - s u p p r e s s e d rabbits. We have r o u t i n e l y i n d u c e d a l l o t y p e s u p p r e s s i o n i n r a b b i t s w h i c h were h e t e r o z y g o u s a t t h e b (kappa c h a i n ) l o c u s b y i m m u n i z i n g homozygous f e m a l e s a g a i n s t t h e b l o c u s a l l o t y p e o f t h e i r p r o s p e c t i v e homozygous mates.
Most
o f t h e p r o g e n y o b t a i n e d b y t h i s method have been c o m p l e t e l y s u p p r e s s e d i n terms o f t h e p a t e r n a l t y p e when f i r s t t e s t e d a t w e a n i n g age ( 8 weeks), u s i n g a t e s t w h i c h d e t e c t s as l i t t l e as 1 pg o f a g i v e n a l l o t y p i c s p e c i f i c i t y (23). T a b l e I l i s t s t h e a l l o t y p i c c o m b i n a t i o n s we have u t i l i z e d t o b r e e d the rabbits f o r our studies.
Data summarized i n T a b l e I 1 show t h a t , i n
each i n s t a n c e , s p l e e n c e l l s t a k e n f r o m an a l l o t y p e - s u p p r e s s e d r a b b i t formed n e u t r a l i z i n g a n t i b o d i e s w h i c h were marked w i t h t h e m a t e r n a l (non-
234
TABLE I Breeding Schemes f o r A l l o t y p e Suppression
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Dam -
Sire
Progeny
b5, a n t i - b 4
b4
( b 4 ) b5
b6, a n t i - b 4
b4
(b4)b6
b6, a n t i - b 5
b5
( b 5 ) b6
b5, a n t i - b 6
b6
b5(b6)
b4, a n t i - b 5
b5
b4(b5)
b4, a n t i - b 6
b6
b4( b6)
The suppressed t y p e o f t h e o f f s p r i n g i s shown i n parentheses.
suppressed) a l l o t y p e o n l y , when t h e y were s t i m u l a t e d w i t h t h e s o l u b l e T2 a n t i g e n (S-T2) i n c u l t u r e .
C e l l s o f normal h e t e r o z y g o t e s formed a n t i -
bodies o f both a l l o t y p i c s p e c i f i c i t i e s encoded by t h e i r genotype.
I n another s e r i e s o f experiments spleen c e l l s o f (b4)b5 (b4-suppressed b4b5) r a b b i t s were
exposed t o
anti-b5
serum
(made i n b4 r a b b i t s )
d u r i n g t h e e n t i r e c u l t u r e p e r i o d o f 4 t o 5 days.
As observed p r e v i o u s l y
when normal b4b5 spleen were c u l t u r e d i n t h e presence o f a : i t i - b 5 , a marked i n h i b i t i o n o f b5 a n t i - T 2 p r o d u c t i o n ( 2 1 ) .
t h e r e was
Surprisingly, i n h i b i t i o n
o f t h e non-suppressed a l l o t y p e ( b 5 ) was accompanied by t h e appearance o f h i g h l y s i g n i f i c a n t t i t e r s c o n t r i b u t e d by b4 a n t i - T 2 ( 2 3 ) .
The d i s t r i b u t i o n
o f a l l o t y p i c s p e c i f i c i t i e s between b4 and b5 a n t i - T 2 a c t i v i t i e s o b t a i n e d under these c o n d i t i o n s o f t e n resembled t h a t formed by normal b4b5 spleen c e l l s (Figure 1).
These r e s u l t s p r o v i d e d d i r e c t evidence f o r t h e
e x i s t e n c e o f an a c t i v e suppression mechanism which c o n t i n u e d t o e x e r t i t s e f f e c t i n v i t r o on c e l l s p o t e n t i a l l y capable o f making t h e suppressed a l l o t y p e , and which c o u l d seemingly be overcome by suppressing t h e a c t i v i t i e s of c e l l s a s s o c i a t e d w i t h p r o d u c t i o n o f t h e non-suppressed I g t y p e .
ANTIBODIES EFFECT ON SYNTHETIC CAPACITY
235
TABLE I 1 A l l o t y p e D i s t r i b u t i o n o f T2 N e u t r a l i z i n g A n t i b o d i e s Made i n Spleen C e l l - C u l t u r e s f r o m Normal and A j j o t y p e - S u p p r e s s e d R a b b i t s A l l o t y p e ( s ) o f Spleen Donor
Grolrp
R e c i p r o c a l o f 302 N e u t r a l i z a t i o n T i t e r * b4 a n t i - T 2 b5 a n t i - T Z b6 a n t i - T 2 ~-
b4b4
64
