UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The effect of continual antigenic stimulation on the immune system of mice McMaster, William Robert 1976

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1976_A6_7 M24.pdf [ 1.9MB ]
Metadata
JSON: 831-1.0100124.json
JSON-LD: 831-1.0100124-ld.json
RDF/XML (Pretty): 831-1.0100124-rdf.xml
RDF/JSON: 831-1.0100124-rdf.json
Turtle: 831-1.0100124-turtle.txt
N-Triples: 831-1.0100124-rdf-ntriples.txt
Original Record: 831-1.0100124-source.json
Full Text
831-1.0100124-fulltext.txt
Citation
831-1.0100124.ris

Full Text

THE EFFECT OF CONTINUAL ANTIGENIC  STIMULATION  ON THE IMMUNE SYSTEM OF MICE by WILLIAM ROBERT McMASTER B.Sc. U n i v e r s i t y o f B r i t i s h Columbia, 1973  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREEMNTS FOR THE DEGREE OF MASTER OF SCIENCE  I n t h e Department of Microbiology  We a c c e p t t h i s t h e s i s as c o n f o r m i n g to t h e r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA F e b r u a r y , 1976  In p r e s e n t i n g t h i s t h e s i s  in p a r t i a l f u l f i l m e n t o f the requirements  for  an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, 1 agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and I f u r t h e r agree t h a t p e r m i s s i o n  for e x t e n s i v e copying of t h i s  study. thesis  f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department or by h i s  representatives.  It  i s understood that copying o r p u b l i c a t i o n  o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my written  permission.  Department o f  /VA^  bio fog  y /  The U n i v e r s i t y o f B r i t i s h Columbia 2075 W e s b r o o k P l a c e V a n c o u v e r , Canada V6T 1W5  Date  Hc^/  ?X  , /  7 %  ii ABSTRACT The e f f e c t o f c o n t i n u a l a n t i g e n i c s t i m u l a t i o n on t h e immune system o f mice was s t u d i e d u s i n g two d i f f e r e n t e x p e r i m e n t a l approaches.  A GVHR was  induced i n F i mice by t h e i n j e c t i o n o f p a r e n t a l s p l e e n c e l l s a t weekly intervals.  S e v e r a l weeks l a t e r t h e s p l e e n c e l l s o f mice u n d e r g o i n g a GVHR  were shown t o be immunosuppressed  a s t h e i r i n v i t r o r e s p o n s e s t o t h e mitogens  Con A and LPS were s u b s t a n c i a l l y lower than c o n t r o l a n i m a l s . t h e s e t r e a t e d mice was a l s o immunosuppressive  The serum from  t o normal s p l e e n c e l l s .  p r o l i f e r a t i v e response t o Con A and a l l o g e n e i c c e l l s o f n o r m a l :  The  syngeneic,  a l l o g e n e i c , and p a r e n t a l s p l e e n c e l l s was 90% suppressed when GVH serum was added i n comparison t o t h e a d d i t i o n o f normal serum. a n t i b o d y response t o a T dependant  S i m i l a r l y , the i n v i t r o  a n t i g e n was i m p a i r e d ; however, t h e a n t i b o d y  response t o a T independant a n t i g e n was n o t i m p a i r e d .  These r e s u l t s  i n d i c a t e t h a t T c e l l f u n c t i o n s a r e more s e n s i t i v e than a r e B c e l l f u n c t i o n t o immunosuppressive  f a c t o r s i n t h e serum o f mice u n d e r g o i n g a GVHR.  The serum was f r a c t i o n a t e d by g e l f i l t r a t i o n on a B i o - G e l P-200 column. The i n h i b i t o r y m a t e r i a l i n GVH serum e l u t e d i n t h e immunoglobulin f r a c t i o n o f serum which i n d i c a t e t h a t i t has a m o l e c u l a r weight o f 150,000 o r g r e a t e r . The second approach s t u d i e d i n v o l v e d c o n t i n u a l a l l o g e n e i c  stimulation.  P a r e n t a l t y p e mice were i n j e c t e d a t f i v e day i n t e r v a l s w i t h F i s p l e e n c e l l s i n o r d e r t o induce a HVG r e a c t i o n .  After several i n j e c t i o n s the spleen c e l l s  from t h e s e mice were t e s t e d i n v i t r o .  The s p l e e n c e l l s from HVG mice r e s p o n d e d  the same as normal s p l e e n c e l l s t o the mitogens Con A and LPS. c e l l s from HVG mice showed an enhanced t o normal s p l e e n c e l l s .  The s p l e e n  i n v i t r o a n t i b o d y response a s compared  T h i s enhancement was a t t r i b u t e d t o t h e a l l o g e n e i c  effect. T h i s s e r i e s o f e x p e r i m e n t s have shown t h a t t h e i n d u c t i o n o f a GVHR i n mice can l a t e r l e a d t o immunosuppression  and p r o d u c t i o n o f immunosuppressive  iii f a c t o r s i n t h e serum o f t h e s e mice.  The i n d u c t i o n o f a s h o r t term HVG  r e a c t i o n has no adverse e f f e c t s on t h e immune system except f o r enhancing an a n t i b o d y response.  I t i s p o s s i b l e t h a t a more p r o l o n g e d HVG r e a c t i o n would  p a r a l l e l t h e immunosuppression  observed i n mice u n d e r g o i n g a GVHR.  iv  TABLE OF CONTENTS Page INTRODUCTION  1  I.  The C e l l s which I n i t i a t e  a GVHR ........ . .  ..  >. 2  II.  F a t e o f t h e Host  III.  The E f f e c t o f GVHR Upon t h e Immune R e s p o n s i v e n e s s o f t h e H o s t . 4  IV.  Immunosuppression  V.  Objective of the Thesis  2  i n Tumour B e a r i n g A n i m a l s  5 6  MATERIAL AND METHODS  8  I.  Animals  8  II.  I n d u c t i o n o f GVHD  8  III.  Induction of Continual Allogeneic Stimulation  IV.  C o l l e c t i o n and P r e p a r a t i o n o f Serum  V.  I n V i t r o Mitogen S t i m u l a t i o n  9  VI.  P r e p a r a t i o n o f DNP-LPS  9  VII.  In V i t r o Antibody Production  10  VIII.  Serum F r a c t i o n a t i o n  10  .  .  RESULTS  8 .8  II  I.  Immune Responses o f A n i m a l s Undergoing GVHD  11  II.  Immune Responses o f A n i m a l s Undergoing C o n t i n u a l A l l o g n e i c S t i m u l a t i o n (HVG)  11  E f f e c t s o f Serum from Normal and GVH M i c e on t h e Immune Responses o f Normal Lymphocytes  15  IV.  G e l F i l t r a t i o n o f Serum  19  V.  Assay o f F r a c t i o n a t e d Serum  III.  . . .  19  DISCUSSION  23  BIBLIOGRAPHY  26  V  LIST OF TABLES Page Table I  Table I I  I n V i t r o Immune Responses o f Spleen C e l l s HVG Mice  from  E f f e c t o f Serum from Normal and GVH Mice on the I n V i t r o P r i m a r y PCC Response  14  20  vi LIST OF FIGURES Page Figure 1.  K i n e t i c s o f m i t o g e n s t i m u l a t i o n s o f c o n t r o l and GVH mice  12  2.  Dose response o f Con A and LPS  13  3.  S t i m u l a t i o n o f normal s p l e e n c e l l s by Con A i n t h e p r e s e n c e o f mouse s e r u m . . . . . . . .  16  4.  V i a b i l i t y o f n o r m a l s p l e e n s c u l t u r e d w i t h mouse serum  17  5.  I n h i b i t i o n o f t h e MLC by GVH serum  18  6.  E l u t i o n p r o f i l e o f normal and GVH serum  21  7.  A s s a y o f f r a c t i o n a t e d serum  22  vii  ACKNOWLEDGEMENT  I would l i k e t o thank Dr. J u l i a L e v y f o r h e r many s u g g e s t i o n s , h e l p f u l c r i t i c i s m s and h e r i n v a l u a b l e encouragement of t h i s t h e s i s .  d u r i n g t h e r e s e a r c h and w r i t i n g  viii ABBREVIATIONS  Abbreviations  u s e d . i n t h i s t h e s i s GVHD, g r a f t - v s - h o s t d i s e a s e ; GVHR,  g r a f t - v s - h o s t r e a c t i o n ; GVH mice; mice undergoing a GVHR, GVH serum, serum from GVH mice; HCG, h o s t - v s - g r a f t ; HVG m i c e , mice u n d e r g o i n g a HVG r e a c t i o n , PHA, p h y t o h e m a g g l u t i n i n , PBS, p h o s p h a t e - b u f f e r e d s a l i n e ; FGS, f e t a l  calf  serum; ConA, c o n c a n a v a l i n A; LPS, b a c t e r i a l l i p o p o l y s a c c h a r i d e ; PFC, plaque f o r m i n g c e l l s ; DNP-LPS, d i n i t r o p h e n y l a t e d LPS, SRBC, sheep r e d b l o o d MLC, mixed lymphocyte  c u l t u r e ; H-TdR, t r i t i a t e d 3  thymidine.  cells;  1 INTRODUCTION The i n t e n s i t y and d u r a t i o n o f an immune response i s c o n t r o l l e d by s e v e r a l r e g u l a t o r y mechanisms.  An immune response i s n o r m a l l y i n i t i a t e d when immuno-  competent c e l l s a r e c o n f r o n t e d w i t h an a n t i g e n .  Once t h e a n t i g e n i s removed  t h e immune response u s u a l l y s u b s i d e s , i m p l y i n g t h a t c o n t i n u a l a n t i g e n i c s t i m u l a t i o n i s n e c e s s a r y f o r a c o n t i n u a l immune r e s p o n s e .  Control of t h i s  l e v e l i s t h e r e f o r e m a i n t a i n e d by t h e source o f a n t i g e n a v a i l a b l e t o t h e lympho i d  eel1s.  S u p p r e s s i o n o f an a n t i b o d y response by p a s s i v e i m m u n i z a t i o n has been observed f o r many a n t i g e n s (Rowely, 1973).  R e l a t i v e l y s m a l l amounts o f  s p e c i f i c a n t i b o d y i f g i v e n w i t h i n t w e n t y - f o u r hours a f t e r i m m u n i z a t i o n w i l l s p e c i f i c a l l y suppress t h e a n t i b o d y response t o t h a t a n t i g e n (Rowely, 1973). T h i s mechanism has been c a l l e d a n t i b o d y feedback i n h i b i t i o n and has a l s o been shown t o o p e r a t e i n v i v o under normal c o n d i t i o n s ( G r a f , 1967). There i s a l s o e v i d e n c e o f a c e l l u l a r c o n t r o l system.  Macrophages can  modulate t h e immune response by p r o c e s s i n g and p r e s e n t i n g a n t i g e n t o o t h e r lymphoid c e l l s .  C e r t a i n thymus-dependant lymphocytes ( s u p p r e s s o r T c e l l s )  can d i r e c t l y e f f e c t t h e p r o d u c t i o n o f a n t i b o d y by thymus independant (B c e l l s ) ( K a t z , 1972).  cells  Under normal p h y s i o l o g i c a l c o n d i t i o n s an immune  system i s not c o n f r o n t e d w i t h a c h r o n i c s o u r c e o f a n t i g e n and t h e r e f o r e a l l the c o n t r o l mechanisms a c t t o r e g u l a t e an e f f e c t i v e immune r e s p o n s e . One model system i n w h i c h immunocompetent c e l l s a r e c o n f r o n t e d w i t h a c o n t i n u a l source o f a n t i g e n i s t h e g r a f t v e r s e s h o s t r e a c t i o n (GVHR).  A  GVHR i s induced by i n j e c t i o n o f lymphoid c e l l s , w h i c h a r e c a p a b l e o f engaging i n an immune r e s p o n s e , i n t o a h o s t w h i c h p o s s e s s a n t i g e n s d i s t i n c t from t h e injected c e l l s .  The h o s t must be i n c a p a b l e o f mounting an immune response  against the grafted c e l l s i n order f o r the grafted c e l l s to survive. s i m p l e s t system t o s t u d y i s t o i n j e c t p a r e n t a l  lymphoid c e l l s i n t o a  The  2 genetically tolerant F i hybrid host. The term GVHR r e f e r s t o t h e i m m u n o l o g i c a l response o f t h e donor lymphoid c e l l s t o t h e f o r e i g n h i s t o c o m p a t i b i l i t y a n t i g e n s o f the h o s t .  The term g r a f t  v e r s u s h o s t d i s e a s e (GVHD) r e f e r s t o t h e complex syndrome which r e s u l t s i n t h e h o s t as a r e s u l t o f t h e GVHR ( E l k i n s , 1971). The C e l l s which I n i t i a t e a GVHR I t has been shown i n a number o f e x p e r i m e n t s t h a t T lymphocytes a r e t h e c e l l s r e s p o n s i b l e f o r a GVHR.  Treatment o f normal s p l e e n c e l l s w i t h  anti-  t h e t a serum and complement, w h i c h k i l l s t h e T c e l l p o p u l a t i o n , w i l l r e n d e r t h e c e l l s i n c a p a b l e o f i n i t i a t i n g a GVHR ( L o n a i , 1973) .  S i m i l a r l y lymphoid  cells  o b t a i n e d from a d u l t mice t h a t have undergone n e o n a t a l thymecotomy a r e a l s o i n c a p a b l e o f i n d u c i n g a GVHR ( M i l l e r , 1967). d i r e c t l y i n d u c e GVHR.  Bone marrow c e l l s do n o t  However t h e y c o n t a i n p r e c u r s o r T c e l l s w h i c h a r e  matured by t h e h o s t ' s thymus and t h e n a r e c a p a b l e o f i n d u c i n g a GVHR (Thomas, 1969).  The t h e o r y t h a t bone marrow c e l l s r e q u i r e t h e h o s t ' s thymus i n o r d e r  to induce a GVHR i s s u p p o r t e d by t h e f a c t t h a t bone marrow c e l l s do n o t induce a GVHR i n thymectomized h o s t s , ( T a y l o r , 1963).  More r e c e n t l y i t has been shown  t h a t t h e r e a r e two t y p e s o f T c e l l s w h i c h might be r e q u i r e d t o e l i c i t a GVHR. S y n e r g i s t i c r e s p o n s e s were o b t a i n e d when s m a l l numbers o f c e l l s from lymphoid t i s s u e s t h a t were r i c h i n GVH a c t i v i t y , such as s p l e e n and lymph nodes, were combined w i t h a l a r g e r number o f weekly r e a c t i v e thymus c e l l s .  The degree o f  synergy was dependant upon t h e r a t i o o f t h e two c e l l t y p e s ( C a n t o r , 1970). Fate o f t h e Host The i n d u c t i o n o f a GVHR i n a h o s t a n i m a l l e a d s t o a complex syndrome. Depending on t h e g e n e t i c d i f f e r e n c e s between t h e h o s t and t h e donor, and t h e number o f donor c e l l s i n j e c t e d , v a r i o u s l e s i o n s o c c u r .  With strong genetic  d i f f e r e n c e s t h e GVHD i s c h a r a c t e r i z e d by l o s s o f body w e i g h t ,  splemomegaly,  3  hematomegaly, lymph node a t r o p h y , immunosuppression, e v e n t u a l l y death. syndromes o c c u r .  g l o m e r u l o n e p h r i t i s , and  Depending on t h e s t r a i n s d f i n b r e d mice used, v a r i o u s I n some s t r a i n c o m b i n a t i o n , one i n j e c t i o n o f donor c e l l s  w i l l l e a d t o death w i t h i n t h i r t y days whereas w i t h o t h e r s t r a i n  combinations  a f t e r f o u r e q u i v a l e n t i n j e c t i o n s , t h e a n i m a l s remain h e a l t h y up t o one y e a r a f t e r the l a s t i n j e c t i o n .  Splenomegaly  and immunosuppression  c o n s t a n t f i n d i n g s w i t h almost e v e r y a n i m a l u n d e r g o i n g a GVHR. was assumed t h a t t h e s p l e e n enlargement  a r e e a r l y and Initially i t  r e s u l t e d from t h e g r a f t e d  p r o l i f e r a t i n g i n response t o t h e h o s t a l l o a n t i g e n s (Simonsen,  cells  1957) .  I n v e s t i g a t i o n s u s i n g chromosome markers soon i n d i c a t e d t h a t most o f t h e p r o l i f e r a t i n g c e l l s were o f h o s t o r i g i n ( E l k i n s , 1966). shown t h a t g r a f t e d c e l l s p r e t r e a t e d w i t h mitomycin-C, s y n t h e s i s , can s t i l l  induce splenomegaly  More r e c e n t l y i t was  which i n h i b i t s DNA  i n F! h o s t s p r o v i d e d they were mixed  w i t h F j c e l l s o v e r n i g h t p r i o r t o i n j e c t i o n i n t o h o s t a n i m a l s ( S c o l l a y , 1974) . These a u t h o r s ' e x p l a n a t i o n f o r t h e i r r e s u l t s was t h a t t h e i n j e c t e d c e l l s c a n mount an immune response t o t h e h o s t ' s a l l o a n t i g e n s ( a s f a r as p o s s i b l e w i t h o u t c e l l d i v i s i o n ) and r e l e a s e s o l u b l e f a c t o r s w h i c h non s p e c i f i c a l l y i n d u c e h o s t cell proliferation.  R e g a r d l e s s o f whether t h e i r e x p l a n a t i o n i s t r u e ,  splenomagaly appears t o be t h e r e s u l t o f h o s t c e l l  proliferation.  There has been one r e p o r t i n w h i c h t h e h o s t appeared the g r a f t e d c e l l s .  tomounta response t o  When p a r e n t a l lymphoid c e l l s were i n j e c t e d i n t o an F i h o s t  t h e s e c e l l s produced a n t i b o d y a g a i n s t t h e h o s t ( i f they were B c e l l s ) o r become c y t o t o x i c ( i f t h e y were T c e l l s ) . which t h e F i c e l l s l a c k t h e m s e l v e s .  The p a r e n t a l c e l l s now have  something  This i s the i d i o t y p e of the a n t i - p a r e n t a l  a n t i b o d y and t h e c o r r e s p o n d i n g r e c e p t o r s on t h e p a r e n t a n t i - F i T and B lymphocytes.  I t was shown t h a t a few F i a n i m a l s i n j e c t e d w i t h p a r e n t a l c e l l s  produced an a n t i b o d y t o t h i s i d i o t y p e ( B i n z , 1975).  A t t h i s time i t remains  u n c l e a r what r o l e i n a GVHR t h i s h o s t response may have.  4  The E f f e c t o f GVHR Upon t h e Immune R e s p o n s i v e n e s s o f t h e Host The immune system o f a n i m a l s u n d e r g o i n g a GVHR has been shown t o be s e v e r e l y immunosuppressed.  B c e l l r e s p o n s e s , a s measured by serum a n t i b o d y  l e v e l s and by PFC, t o b o t h T dependant  a n t i g e n s ( D a v i e s , 1970) and T i n -  dependant a n t i g e n ( M o l l e r , 1971) have been shown t o be s u p p r e s s e d .  Allograft  r e j e c t i o n has been found t o be p r o l o n g e d i n a n i m a l s w i t h c h r o n i c GVH, i m p l y i n g t h a t T c e l l f u n c t i o n was suppressed under t h e s e c o n d i t i o n s (Lapp, 1969) . an attempt t o overcome t h e immunosuppression  In  i n GVH m i c e , normal s y n g e n e i c  lymphoid c e l l s were a d o p t i v e l y t r a n s f e r r e d i n t o the h o s t mice  (Claman, 1969) .  T h i s t r a n s f e r d i d n o t r e s t o r e immunocompetence o f "the h o s t i m p l y i n g t h a t t h e donated competent  c e l l s had become immunosuppressed.  I n a n o t h e r system i t was  shown t h a t F i a n i m a l s w h i c h had r e c e i v e d one i n j e c t i o n o f p a r e n t a l c e l l s were r e s i s t a n t t o a second i n j e c t i o n o f p a r e n t a l c e l l s which would n o r m a l l y cause a c u t e GVH i n u n t r e a t e d a n i m a l s ( F i d d , 1966).  R e s i s t a n c e t o t h e second  c h a l l e n g e was t r a n s f e r r e d t o normal F i a n i m a l s by p a r a b i o s i s o r c r o s s c i r c u l a t i o n ( F i e l d , 1967).  T h i s experiment i n d i c a t e d t h a t t h e r e s i s t a n t  s t a t e was due t o e i t h e r i n h i b i t o r y f a c t o r s o r s u p p r e s s o r c e l l s i n t h e b l o o d of  these animals.  There have been a few a t t e m p t s t o demonstrate i n v i v o  t h a t serum from GVH mice c o n t a i n s f a c t o r s w h i c h c a n suppress t h e immune system o f normal a n i m a l s ( G r u s h k a , 1974).  These e x p e r i m e n t s were u n s u c c e s s f u l ,  perhaps due t o i n s u f f i c i e n t amounts o f serum t r a n s f e r r e d .  More r e c e n t l y , i t  was demonstrated t h a t s p l e e n c e l l s from GVH mice when c u l t u r e d i n v i t r o produce a f a c t o r ( s ) w h i c h s u p p r e s s e s t h e response o f normal s p l e e n c e l l s t o PHA  (Phillips,  1975).  In a d d i t i o n t o t h e d e m o n s t r a t i o n o f i n h i b i t o r y f a c t o r s o t h e r workers have r e c e n t l y shown t h a t c e r t a i n c e l l p o p u l a t i o n s from t h e s p l e e n s o f a n i m a l s underg o i n g GVHR c a n suppress an i n v i t r o immune r e s p o n s e .  I t was shown t h a t t h e  adherent c e l l s from t h e s e a n i m a l s , when c u l t u r e d w i t h normal s p l e e n c e l l s  5  would suppress t h e g e n e r a t i o n o f PFC t o t h e T dependant ( E l i e , 1975).  a n t i g e n , SRBC  The adherent c e l l s from t h e s p l e e n s o f a n i m a l s u n d e r g o i n g a  GVHR d i d n o t appear t o be s o l e l y  suppressor c e l l s but served t o f u n c t i o n as  r e g u l a t o r y c e l l s i n t h e i n d u c t i v e phase o f an immune r e s p o n s e .  T h i s was based  on t h e i r r e s u l t s which showed t h a t t h e s e adherent c e l l s c o u l d r e p l a c e normal adherent c e l l s i f added i n c e r t a i n r a t i o s .  They p o s t u l a t e d from t h e i r  in vitro  r e s u l t s t h a t an e x c e s s number o f t h e adherent c e l l s i n the h o s t ' s s p l e e n may be r e s p o n s i b l e f o r t h e observed i n v i v o immunosuppression  o f humoral r e s p o n s e s .  Summarizing t h e s e f i n d i n g s i t appears t h a t t h e immune system o f an a n i m a l u n d e r g o i n g a GVHR may be immunosuppressed  due t o an e x c e s s number o f  s u p p r e s s o r c e l l s w h i c h i n t u r n produce an i n h i b i t o r y the  f u n c t i o n o f o t h e r lymphoid c e l l  Immunosuppression  f a c t o r ( s ) which suppresses  populations.  i n Tumour B e a r i n g A n i m a l s  Another s i t u a t i o n  i n which animals a r e subjected t o excess a n t i g e n a r e  those a n i m a l s w h i c h have a p r o g r e s s i v e l y growing tumour. t h a t immunosuppression  o f t e n accompanies  tumour growth.  I t has been shown Tumour  specific  immunity which i s p r e s e n t d u r i n g e a r l y growth o f t h e tumour has been shown t o d e c r e a s e as t h e tumour s i z e i n c r e a s e s ( D e c k e r s , 1973). along w i t h the decrease i n s p e c i f i c g e n e r a l immunocompetence.  I n some tumour systems,  immunity t h e r e i s a l s o a d e c r e a s e i n  T h i s g e n e r a l immunocompetence has been measured by  l a c k o f a b i l i t y o f s p l e e n c e l l s from tumour b e a r i n g mice t o respond non specifically  t o mitogens (Whitney, 1974), t o mount a response t o a l l o g e n e i c  c e l l s (Haran-Gheru, 1973), and t o produce a n t i b o d y t o a d e f i n e d a n t i g e n ( S m i t h , 1973). There a r e s e v e r a l p o s s i b l e causes o f t h e immunosuppression accompanies tumour growth.  which  P r e v i o u s work h a s shown t h a t serum from mice w i t h  l a r g e tumours i s c a p a b l e o f s u p p r e s s i n g t h e p r o l i f e r a t i v e mouse s p l e e n c e l l s t o mitogens and a l l o g e n e i c c e l l s  response o f normal  (Whitney, 1975).  I t has  6 been found t h a t the m a t e r i a l r e s p o n s i b l e f o r t h i s i n h i b i t i o n e l u t e d w i t h immunoglobulin on Shephadex G-150.  F u r t h e r m o r e , the i n h i b i t o r y f a c t o r c o u l d  be removed on an anti-mouse immunoglobulin immunoadsorbent ( L e v y , 1975) .  These  r e s u l t s i n d i c a t e t h a t the i n h i b i t o r y s u b s t a n c e i s a n t i b o d y - l i k e i n n a t u r e and c o u l d be an a n t i b o d y - a n t i g e n complex o r a r e g u l a t o r y a n t i b o d y . The s p l e e n s , from t h o s e a n i m a l s whose serum i s immunosuppressive, c o n t a i n c e l l s which can s u p p r e s s the response o f normal s p l e e n c e l l s t o mitogens (Pope, 1975).  The r e l a t i o n s h i p between the s u p p r e s s o r c e l l s and the  serum f a c t o r i s u n c l e a r ; however one might s p e c u l a t e t h a t t h e serum f a c t o r was a p r o d u c t of t h e s u p p r e s s o r c e l l s i n response t o e x c e s s a n t i g e n .  Another  s o u r c e of t h e serum f a c t o r w h i c h cannot be i g n o r e d i s t h a t i t c o u l d be a p r o d u c t from t h e tumour c e l l s .  T h i s i s u n l i k e l y , since these suppressive  s u b s t a n c e s can be removed on immunoadsorbent columns p r e p a r e d w i t h  anti-mouse  immunoglobulin. O b j e c t i v e of the T h e s i s The o b j e c t i v e o f t h i s t h e s i s was t o s t u d y the e f f e c t s on the immune system of c o n t i n u a l a n t i g e n i c s t i m u l a t i o n s . (1)  Two model systems were used:  A GVHR was i n d u c e d i n F i a n i m a l s by the i n j e c t i o n of p a r e n t a l s p l e e n cells.  T h i s enabled the g r a f t e d c e l l s t o be i n c o n s t a n t c o n t a c t w i t h  a l l o g e n e i c a n t i g e n w h i l e the h o s t was g e n e t i c a l l y t o l e r a n t t o t h e grafted (2)  cells.  A n i m a l s (mice) were s u b j e c t e d t o c o n t i n u a l a l l o g e n e i c s t i m u l a t i o n by repeated a d m i n i s t r a t i o n of semi-allogeneic ( F i ) spleen c e l l s .  This  i n i t i a t e d a one way "Host v s . G r a f t " response s i n c e the i n j e c t e d  cells  were t o l e r a n t ' t o t h e h o s t . The lymphoid c e l l s o f t h e s e a n i m a l s were t h e n assayed by v a r i o u s i n v i t r o measurements o f immunocompetance.  I t w i l l be shown t h a t c o n t i n u a l a l l o g e n e i c  s t i m u l a t i o n s has no adverse e f f e c t s on the immune system o f mice w h i l e the  7  i n d u c t i o n of a GVHR i n mice l e a d s t o immunosuppression.  8  MATERIALS AND METHODS  Animals BALB/c, DBA/2J, CBA/J, C57BL/6J,  and C57BL/6 X DBA/2J F i (B6D2 ¥ ) x  Female mice ( J a c k s o n L a b o r a t o r y , Bar Harbour, Maine) aged 2-4 months were used throughout t h i s s t u d y . I n d u c t i o n o f GVHD GVHD was induced i n F i a n i m a l s by f o u r weekly i n t r a p e r i t o n e a l of 50 x 1 0 p a r e n t a l (DBA/2J) s p l e e n c e l l s . 6  GVH mice.  injections  These mice w i l l be r e f e r r e d t o as  C o n t r o l mice c o n s i s t e d o f F i a n i m a l s i n j e c t e d as above w i t h 50 x 1 0  Fx s p l e e n c e l l s .  6  M i c e u n d e r g o i n g GVHD induced i n t h i s way were c l i n i c a l l y w e l l ,  a l t h o u g h s p l e e n s i z e a t t i m e o f s a c r i f i c e was m o d e r a t e l y e n l a r g e d (2-3 f o l d ' i n c r e a s e i n weight over normal  controls).  Induction of Continual Allogeneic  Stimulation  C o n t i n u a l a l l o g e n e i c s t i m u l a t i o n was i n d u c e d i n DBA/2J mice by 7 i n j e c t i o n s , a t 5 day i n t e r v a l s o f 20 x 1 0 F i s p l e e n c e l l s . 6  These mice w i l l  be r e f e r r e d t o as HVG mice. C o l l e c t i o n and P r e p a r a t i o n o f Serum B l o o d was c o l l e c t e d from the r e t r o - o r b i t a l s i n u s o f n o r m a l , c o n t r o l , and GVH mice 4 and 6 weeks a f t e r t h e l a s t i n j e c t i o n o f c e l l s .  The b l o o d was  a l l o w e d t o c l o t f o r 2 hours a t 4?C a f t e r w h i c h t h e serum was s e p a r a t e d and p o o l e d i n t o t h e t h r e e groups.  I t was heat i n a c t i v a t e d a t 56°C f o r 30 m i n ,  absorbed w i t h SRBC f o r 30 min and d i a l y z e d a g a i n s t RPMI 1640 media f o r 24 h o u r s . F o l l o w i n g d i a l y s i s t h e serum was u l t r a c e n t r i f u g e d a t 100,000 x g f o r 3 h o u r s , f i l t e r e d t h r o u g h a m i l l i p o r e 0.22 u f i l t e r and s t o r e d a t -20°C.  9 In V i t r o Mitogen S t i m u l a t i o n s Spleen c e l l s were prepared  i n PBS + 5% FCS.  The c e l l s were c e n t r i f u g e d  at 2000 x g f o r 5 m i n , resuspended i n PBS + 5% FCS, and t h e v i a b i l i t y determined by t r y p a n b l u e e x c l u s i o n .  D e s i r e d number o f c e l l s were c e n t r i f u g e d and r e s u s -  pended i n RPMI 1640 medium and d i s p e n s e d C h e m i c a l Co.).  into m i c r o t i t e r plates (Linbro  Each w e l l c o n t a i n e d 5 x 1 0 v i a b l e s p l e e n c e l l s i n a f i n a l 5  volume o f 0.25 ml medium w i t h FCS a t a f i n a l c o n c e n t r a t i o n o f 2%, and a p p r o p r i a t e c o n c e n t r a t i o n s o f Con A, LPS, o r mouse serum.  The c u l t u r e s were  i n c u b a t e d a t 37°C i n a h u m i d i f i e d atmosphere o f 95% a i r - 5% CO2 f o r 1-4 days as i n d i c a t e d i n each e x p e r i m e n t .  E i g h t e e n h o u r s b e f o r e t h e c e l l s were h a r v e s t e d  1.0 u C i o f H-Thymidine ( s p . a c t . 5.0 Ci/mmol, New England N u c l e a r , 3  Canada) was added t o each w e l l .  The c e l l s were h a r v e s t e d  f i l t e r p a p e r , d r i e d , and t h e r a d i o a c t i v i t y determined.  onto g l a s s  5  fiber  Two way MLC were  performed as d e s c r i b e d above by c u l t u r i n g t o g e t h e r 2.5 x 1 0 and 2.5 x 1 0  Montreal,  s  CBA/J s p l e e n  cells  BALB/c s p l e e n c e l l s and h a r v e s t i n g a f t e r 4 days i n c u b a t i o n .  were r u n i n t r i p l i c a t e , and t h e r e s u l t s expressed  as the mean v a l u e .  Tests  The  standard e r r o r i n a l l i n s t a n c e s was always < 10%. P r e p a r a t i o n o f DNP-LPS LPS, E. c o l i 055:B5, ( D i f c o L a b r a t o r i e s ) was d i s s o l v e d t o a c o n c e n t r a t i o n of 20 mg/ml i n b o r a t e - s a l i n e (pH 8.5).  2,4-dinitrobenzene-sulfonic acid  (Eastman Kodak Co.) and sodium c a r b o n a t e ( F i s h e r C h e m i c a l s ) were then each added t o a f i n a l c o n c e n t r a t i o n o f 20 mg/ml. hours a t room temperature and u n r e a c t e d  The r e a c t i o n was s t i r r e d  f o r 24  r e a g e n t s were removed by e x h a u s t i v e  d i a l y s i s a g a i n s t b o r a t e - s a l i n e f o r 48 h o u r s .  The DNP-LPS was t h e n s t e r i l i z e d  by m i l l i p o r e f i l t r a t i o n and s t o r e d a t -20°C.  I t has been shown t h a t DNP-LPS  c u l t u r e d w i t h normal s p l e e n c e l l s a t sub m i t o g e n i c s p e c i f i c a n t i b o d y response w i t h o u t response);  ( J a c o b s , 1975) .  doses i n v o k e s a DNP  t h e h e l p o f T lymphocytes (T independant  10 In V i t r o Antibody  Production  S p l e e n c e l l s were p r e p a r e d i n RPMI 1640 + 5% FCS b u f f e r e d w i t h 35 mM Hepes b u f f e r .  C e l l s were c u l t u r e d a t a c o n c e n t r a t i o n o f 10 x 1 0  6  i n 35 mm  p e t r i p l a t e s ( F a l c o n #3301) c o n t a i n i n g 2.0 ml RPMI 1640 media supplemented w i t h 10% FCS ( M i c r o b i o l o g i c a l A s s o c i a t e s #84557), 5 x 1 0 ~ M 2 - m e r c a p t o e t h a n o l , 5  and v a r y i n g p e r c e n t a g e s o f mouse serum. SRBC o r 0.10 Ug/ml DNP-LPS. p l a t f o r m f o r 4 days.  Antigens  used were e i t h e r 5 x 1 0  C u l t u r e s were i n c u b a t e d  6  a t 37°C on a r o c k i n g  The number o f d i r e c t PFC was d e t e r m i n e d w i t h a  m i c r o s c o p e s l i d e a s s a y (Cunninghamm, 1968).  DNP p l a q u e s were d e t e r m i n e d  SRBC coated w i t h d i n i t r o p h e n y l a t e d r a b b i t a n t i - S R B C - F a b  1  (Strausbach,  using  1970).  S p e c i f i c anti-DNP p l a q u e s were enumerated by s u b t r a c t i n g background SRBC p l a q u e s from t h e t o t a l number o b t a i n e d . Serum F r a c t i o n a t i o n Samples o f b o t h n o r m a l F i and GVH serum were chromatographed on an a c e n d i n g B i o - G e l P-200 column (2.6 cm x 90 cm) u s i n g b o r a t e - s a l i n e (pH 8.5) as t h e e l u t i n g b u f f e r .  The sample volumes were 1 m l , e l u t i o n f l o w r a t e was  10 ml/hour and 2.0 ml f r a c t i o n s c o l l e c t e d . e l u t i n g from t h e column were pooled s a t u r a t e d ammonium s u l f a t e .  Peaks o f 280 nm a b s o r b i n g  and c o n c e n t r a t e d  by p r e c i p i t a t i o n w i t h 50%  The p r e c i p i t a t e s were k e p t a t 4°C f o r 18 hours  then c e n t r i f u g e d a t 18,000 x g f o r 20 min.  The p e l l e t s were d i s s o l v e d i n  b o r a t e - s a l i n e and d i a l y z e d a g a i n s t t h e same.  The d i a l y z e d m a t e r i a l s were  made up t o t h e o r i g i n a l volumes o f t h e serum sample, f i l t e r e d and  material  sterilized,  s t o r e d a t -20°C. To a s s a y f o r i n h i b i t o r y a c t i o n , . t h e a p p r o p r i a t e f r a c t i o n s were d i l u t e d i n  1640  medium and added t o s p l e e n c e l l c u l t u r e s e x a c t l y as d e s c r i b e d  above.  11 RESULTS Immune Responses o f A n i m a l s Undergoing GVHD The s p l e e n c e l l s of c o n t r o l and GVH mice, o b t a i n e d 8 weeks a f t e r the l a s t i n j e c t i o n of c e l l s , were s t i m u l a t e d i n v i t r o w i t h the T c e l l mitogen Con and t h e B c e l l mitogen LPS.  A,  B o t h c o n t r o l and GVH c e l l s f o l l o w s i m i l a r k i n e t i c s  shown i n F i g . 1.as t h e maximal H-Thymidine 3  each mitogen and each c e l l t y p e .  i n c o r p o r a t i o n o c c u r r e d on day 3 f o r  The maximum Con A response o f c e l l s from  mice was 40% of t h e maximum response o f c e l l s from c o n t r o l mice.  The  GVH  LPS  response of GVH c e l l s was somewhat l e s s s e v e r e l y s u p p r e s s e d , b e i n g 73% o f the response of c o n t r o l c e l l s .  The s u p p r e s s i o n of GVH c e l l s i s not due t o a  d i f f e r e n c e i n dose r e s p o n s e .  C e l l s from c o n t r o l and GVH mice respond  o p t i m a l l y t o t h e same c o n c e n t r a t i o n of Con A and t o the same c o n c e n t r a t i o n s of LPS ( F i g . 2 ) .  P r e l i m i n a r y d a t a showed t h a t t h e s e dose responses a r e the same_  r e g a r d l e s s o f t h e day on w h i c h the c e l l s were h a r v e s t e d . Immune Responses o f A n i m a l s Undergoing C o n t i n u a l A l l o g e n e i c S t i m u l a t i o n  (HVG)  The s p l e e n c e l l s o f normal and HVG m i c e , o b t a i n e d 4 days a f t e r the l a s t i n j e c t i o n o f F i c e l l s , were s t i m u l a t e d i n v i t r o w i t h Con A and LPS. r e s u l t s a r e shown i n T a b l e I .  These  C e l l s from the HVG a n i m a l s showed a h i g h e r  response t o b o t h Con A and LPS and a l s o had a h i g h e r spontaneous  3  H-Thymidine  uptake. Spleen c e l l s from normal and HVG mice were a l s o t e s t e d f o r an i n v i t r o a n t i b o d y response t o SRBC.  These r e s u l t s a r e a l s o shown i n T a b l e I .  The  cells  from HVG mice showed a much h i g h e r a n t i b o d y r e s p o n s e , b e i n g almost t w i c e as h i g h as the response o f normal c e l l s .  A  .  0  1  2  3  DAYS FIGURE 1.  K i n e t i c s o f mitogen s t i m u l a t i o n s o f s p l e e n c e l l s  from c o n t r o l mice  (data  from 4 m i c e ) , • - — • ; and o f s p l e e n c e l l s , from GVH mice (data from 9 m i c e ) , 1  A.  (A) c e l l s c u l t u r e d w i t h 20 Ug/ml LPS.  Ug/ml Con A.  (B) c e l l s c u l t u r e d w i t h  2.0  13  o  i  O  2 CL U  z  o K-  <  oc O  a. oc  O u z z Q  > x  rI  n  X  1 CON  2 A (ug/ml)  4  FIGURE 2. Dose response o f mitogen s t i m u l a t i o n s o f s p l e e n c e l l s from c o n t r o l mice ( d a t a from 4 m i c e ) , A  i.  and o f s p l e e n c e l l s from GVH mice ( d a t a from 9 m i c e ) ,  (A) LPS dose response  (B) Con A dose r e s p o n s e .  IN VITRO IMMUNE RESPONSES OF SPLEEN CELLS FROM HVG MICE Spleen C e l l s  Mitogen  From  3  HVG M i c e  Antibody  H-TdR Uptake (cpm)  Medium  Normal M i c e  Responses  LPS  8,720  53,177  17,789  61,594  Con A  82,093. 125,245  Responses  Pfc/culture Medium  SRBC  226  4,146  526  8,737  Table I  The i n v i t r o mitogen and a n t i b o d y r e s p o n s e s o f s p l e e n c e l l s from HVG mice.  13  E f f e c t s o f Serum from Normal and GVH M i c e o f the Immune Responses of Normal Lymphocytes The f o l l o w i n g e x p e r i m e n t s were d e s i g n e d t o t e s t whether GVH immunosuppressive  t o lymphoid c e l l s from normal mice.  serum i s  Serum o b t a i n e d  n o r m a l , c o n t r o l , o r GVH mice was c u l t u r e d w i t h normal F i , o r DBA/2J c e l l s and v a r i o u s amounts o f Con A.  from spleen  The r e s u l t s o f t h e s e e x p e r i m e n t s a r e  shown i n F i g . 3. The o p t i m a l  response t o Con A of normal F i s p l e e n  c e l l s was  suppressed  by the a d d i t i o n of GVH serum as compared t o the a d d i t i o n s of normal serum. The response of c e l l s c u l t u r e d w i t h 2% GVH c e l l s c u l t u r e d w i t h 2% normal serum.  serum was 30% of the response of  Increasing  serum t o 4% was even more immunosuppressive  as t h o s e c e l l s used t o i n d u c e the GVH. c e l l s c u l t u r e d w i t h 4% GVH w i t h 4% normal serum.  of  GVH  as the response was 9% of the  response o f c e l l s c u l t u r e d w i t h 4% normal serum. the Con A response of normal DBA/2J s p l e e n  the c o n c e n t r a t i o n  GVH  serum a l s o  inhibited  c e l l s w h i c h a r e the same genotype  The o p t i m a l  Con A response o f t h e s e  serum was 60% of the response of c e l l s  I n t h e s e and o t h e r e x p e r i m e n t s c u l t u r e s  cultured  containing  normal serum gave the same r e s u l t s as c u l t u r e s c o n t a i n i n g c o n t r o l serum, t h e r e f o r e the c o n t r o l i n a l l f u t u r e e x p e r i m e n t s i s termed normal serum. Neither  o f the mouse s e r a were c y t o t o x i c t o normal F i s p l e e n c e l l s , as  the v i a b i l i t i e s of c e l l s c u l t u r e d w i t h normal or GVH v i a b i l i t i e s of c e l l s c u l t u r e d a l o n e w i t h FCS GVH  serum p a r a l l e l e d the  (Fig. 4).  serum was a l s o t e s t e d f o r i t s a b i l i t y t o i n h i b i t the mixed  r e a c t i o n between a l l o g e n e i c c e l l s .  lymphocyte  These r e s u l t s a r e shown i n F i g . 5 and a r e  expressed as a p e r c e n t a g e response of c u l t u r e s c o n t a i n i n g normal mouse serum. As i s e v i d e n t , Further  a t 4% GVH  serum c o n c e n t r a t i o n  the response i s 93% s u p p r e s s e d .  e x p e r i m e n t s were c a r r i e d out t o determine whether GVH  i n h i b i t e d T c e l l f u n c t i o n and/or B c e l l f u n c t i o n , by a s s a y i n g  serum  f o r the i n  2  4 CON  8 A  16  32  64  (ug/ml)  FIGURE 3. Stimulation of normal spleen c e l l s by various amounts of Con A i n the presense of serum from normal mice, • — - • ; or i n the presence of serum from GVH mice, L  A.  (A) normal B6D2 F i spleen c e l l s cultured with 2% mouse serum.  (B) normal B6D2 Fi spleen c e l l s cultured with 4% mouse serum. spleen c e l l s cultured with 4% mouse serum.  (C) normal DBA/2J  0  1  2  3  4  DAYS  FIGURE 4.  V i a b i l i t y o f normal B6D2 F i s p l e e n c e l l s c u l t u r e d w i t h 0% mouse serum, H  B;  c u l t u r e d w i t h 4% serum from normal m i c e , •  serum from GVH m i c e , A  A.  •; and c u l t u r e d w i t h 4%  18  .25  0  .5  1  2  4  SERUM CONCENTRATION %  FIGURE 5. I n h i b i t i o n by serum from GVH mice of the mixed lymphocyte r e a c t i o n between B a l b / c and BCA/J s p l e e n c e l l s .  The r e s u l t s a r e e x p r e s s e d as % of the response  of s p l e e n c e l l s c u l t u r e d w i t h c o r r e s p o n d i n g amounts of serum from normal mice.  19 v i t r o g e n e r a t i o n o f PFC i n i t s p r e s e n c e .  S p l e e n c e l l s from normal F i mice  were c u l t u r e d w i t h v a r i o u s p e r c e n t a g e s o f normal or GVH w i t h e i t h e r the T dependent DNP-LPS.  serum.and s t i m u l a t e d  a n t i g e n , SRBC, or the T independent a n t i g e n ,  These r e s u l t s a r e shown i n T a b l e 2.  A l t h o u g h b o t h normal and  serum i n h i b i t the response t o SRBC, a t e q u a l c o n c e n t r a t i o n s the GVH  GVH  serum i s  always much more s u p p r e s s i v e . The response t o DNP-LPS was not suppressed by any c o n c e n t r a t i o n of GVH  serum assayed b u t showed a somewhat d e p r e s s e d  response w i t h 4% normal serum. Gel  F i l t r a t i o n o f Serum F i g u r e 6 shows t h e e l u t i o n p r o f i l e of normal and GVH  B i o - G e l P-200 column.  serum e l u t e d from a  The f i r s t peak c o r r e s p o n d s t o the immunoglobulin  f r a c t i o n o f serum w h i c h c o n t a i n s m o l e c u l e s h a v i n g a m o l e c u l a r w e i g h t o f a p p r o x i m a t e l y 150,000 o r g r e a t e r .  The second peak c o r r e s p o n d s t o the a l b u m i n  f r a c t i o n o f serum w h i c h c o n t a i n s m o l e c u l e s h a v i n g a m o l e c u l a r w e i g h t s m a l l e r than 100,000.  The f i r s t peak of GVH  serum appears t o have more m a t e r i a l  than does peak 1 of normal serum w h i l e the second peaks appear t o e q u a l amounts of m a t e r i a l . Assay of F r a c t i o n a t e d Serum The P-200 f r a c t i o n s of normal and GVH to  serum were t e s t e d f o r t h e i r  i n h i b i t the p r o l i f e r a t i v e response o f normal F i s p l e e n c e l l s t o Con  These r e s u l t s a r e shown i n F i g u r e 7. from GVH  serum, was immunosuppressive,  ability A.  At h i g h e r c o n c e n t r a t i o n s , f r a c t i o n I i n h i b i t i n g the Con A response by 40% as  compared t o f r a c t i o n I from normal serum.  By e x p r e s s i n g the r e s u l t s as a  p e r c e n t o f response o b t a i n e d w i t h normal f r a c t i o n s i t appears t h a t f r a c t i o n I I from GVH  serum i s s t i m u l a t o r y .  However the t o t a l uptake of  H-thymidine  always h i g h e r i n c o n t r o l c u l t u r e s which d i d not c o n t a i n any mouse serum fractions.  F r a c t i o n I I from GVH  f r a c t i o n I I from normal serum.  serum i s t h e r e f o r e not as s u p p r e s s i v e as  was  20  EFFECT OF SERUM FROM NORMAL AND GVH MICE ON THE IN VITRO PRIMARY PFC RESPONSE  SRBC PFC/CULTURE  DNP PFC/CULTURE  % Serum Added  Normal  0.5  4350 + 665  2750  1.0  4166 + 604  1278 ± 111  2.0  2641 + 634  4.0  695 +  GVH  Normal  269  GVH  5544 + 1053  5048 + 155  5925 +  420  6743 + 283  471 ±  40  6049 +  241  6898 + 641  76 ±  11  3717 + 1115  6357 + 340  69  Table I I The I n f l u e n c e o f serum from normal and GVH mice on t h e development o f PFC i n v i t r o t o SRBC and DNP-LPS.  The response i n u n s t i m u l a t e d c u l t u r e s was always  < 10% o f t h e c o r r e s p o n d i n g s t i m u l a t e d  culture.  21  NORMAL SERUM  1.5  1.0  0.5  < H  1.5  1.0  0.5  10  20  30  kO.  50  60  70 -  FRACTION NUMBER  FIGURE 6. E l u t i o n p r o f i l e o f normal and GVH  serum from a B i o - G e l P-200 column.  22  21  82  4% 7  FINAL  CONCENTRATION  fO  FIGURE 7. E f f e c t of f r a c t i o n I and I I from B i o - G e l P-200 on the Con A response of normal F i s p l e e n c e l l s .  The r e s u l t s a r e e x p r e s s e d as % of the response  o b t a i n e d w i t h an e q u i v a l e n t f r a c t i o n of normal serum i n c u l t u r e w i t h normal 1 vmphoc  23 DISCUSSION  The r e s u l t s shown i n F i g . 1 and 2 demonstrate t h a t t h e s p l e e n c e l l s from GVH mice a r e i m p a i r e d i n t h e i r a b i l i t y t o p r o l i f e r a t e i n v i t r o i n r e s p o n s e t o mitogens.  T c e l l s may be more a f f e c t e d than B c e l l s because t h e Con A  responses a r e more suppressed than the LPS r e s p o n s e s .  These r e s u l t s  correlate  w e l l w i t h numerous i n v i v o s t u d i e s which show t h a t b o t h T c e l l (Lapp, 1969) and B c e l l  ( D a v i e s , 1970 and M o l l e r , 1971) r e s p o n s e s a r e suppressed i n GVH  mice. Recent s t u d i e s on murine l e u k e m i a v i r u s (MuLV), w h i c h i s a c t i v a t e d i n some mice by c h r o n i c GVH d i s e a s e , a l s o showed t h a t t h e s p l e e n c e l l s o f t h e s e mice a r e immunosuppressed  ( P h i l l i p s , 1975).  I t has been r e p o r t e d t h a t i n t h e  mouse s t r a i n c o m b i n a t i o n s used i n t h e p r e s e n t s t u d y , l e s s t h a n 40% o f GVH mice had d e t e c t a b l e l e v e l s o f MuLV ( P h i l l i p s , 1975).  I t i s u n l i k e l y t h a t immuno-  s u p p r e s s i o n observed i n GVH mice i s due t o MuLV because one would e x p e c t , w i t h the s t r a i n c o m b i n a t i o n s used immunosuppressed,  i n t h i s s t u d y , l e s s than 40% of t h e mice t o be  whereas i t was found t h a t e v e r y GVH mouse was  immunosuppressed.  The s p l e e n c e l l s from mice i n j e c t e d w i t h a l l o g e n e i c F i c e l l s show a normal o r i n c r e a s e d i n v i t r o response t o mitogens as compared t o u n t r e a t e d mice.  The i n c r e a s e i n H-Thymidine 3  uptake i n u n s t i m u l a t e d c u l t u r e s of i n j e c t e d  mice i s p r o b a b l y t h e r e s u l t of an ongoing immune response t o t h e . - a l l o g e n e i c  cells.  The i n c r e a s e d mitogen responses may be a r e f l e c t i o n t h a t t h e r e i s a more a c t i v e p o p u l a t i o n o f T and B c e l l s i n t h e s p l e e n s o f t h e s e a n i m a l s . The i n c r e a s e d number o f PFC i n c u l t u r e s from i n j e c t e d mice can be a t t r i b u t e d to the " a l l o g e n e i c e f f e c t . "  T h i s enhancement o f i n v i t r o a n t i b o d y  responses i s t h e r e s u l t o f n o n - s p e c i f i c enhancing f a c t o r s s e c r e t e d by a l l o a c t i v a t e d T c e l l s ( K a t z , 1972). I t becomes apparent t h a t a c o n t i n u a l a l l o g e n e i c s t i m u l a t i o n has no d e l e t e r i o u s e f f e c t s on t h e immune system o f such t r e a t e d mice.  A GVHR does  24 however have some  e f f e c t on t h e immune system o f these i n j e c t e d m i c e .  In t h i s  s i t u a t i o n t h e mice become immunosuppressed, however t h i s may be b e n e f i c i a l i n t h a t t h e g r a f t e d c e l l s a l s o become immunosuppressed and t h e r e f o r e a r e no longer capable o f r e a c t i n g against the host. The mice u n d e r g o i n g a GVHR were n o t immunosuppressed u n t i l s e v e r a l weeks a f t e r t h e i n i t i a t i o n o f t h e GVHR.  Perhaps i f t h e a l l o g e n e i c s t i m u l a t i o n was  c a r r i e d out f o r t h i s same l e n g t h o f t i m e , immunosuppressions would have a l s o occurred. The serum, from a n i m a l s whose s p l e e n c e l l s a r e immunosuppressed due t o GVHD, i s c a p a b l e o f i n h i b i t i n g t h e f u n c t i o n o f normal s y n g e n e i c , s e m i s y n g e n e i c , and a l l o g e n e i c s p l e e n c e l l s .  The s u p p r e s s i v e a b i l i t y o f t h i s serum  cannot be d i r e c t l y a t t r i b u t e d t o i n f e c t i o u s v i r u s because t h e serum was u l t r a c e n t r i f u g e d b e f o r e i t was assayed.  S i m i l a r l y , l o w m o l e c u l a r w e i g h t t o x i c by  p r o d u c t s n o t removed by t h e k i d n e y s w h i c h a r e sometimes damaged by c h r o n i c GVH ( L e w i s , 1968) and s t e r o i d s would have been removed d u r i n g t h e d i a l y s i s a g a i n s t t i s s u e c u l t u r e medium. At t h e c o n c e n t r a t i o n s a s s a y e d , GVH serum appears t o a f f e c t T c e l l f u n c t i o n r a t h e r than B c e l l f u n c t i o n i n t h a t i t i n h i b i t s t h e p r o l i f e r a t i v e response o f normal s p l e e n c e l l s t o Con A, i n h i b i t s t h e mixed lymphocyte r e a c t i o n , and i n h i b i t s t h e a n t i b o d y r e s p o n s e t o a T dependent a n t i g e n b u t not t o a T independent a n t i g e n .  A t h i g h e r serum c o n c e n t r a t i o n s B c e l l  function  might become suppressed i n d i c a t i n g T c e l l s a r e more s e n s i t i v e s t o t h e serum f a c t o r o r e l s e t h e r e may be a n o t h e r f a c t o r a f f e c t i n g B c e l l s .  I t cannot be  r u l e d out a t t h i s time t h a t t h e i n h i b i t o r y m a t e r i a l c o u l d be a f f e c t i n g macrophages w h i c h a r e sometimes n e c e s s a r y f o r t h e g e n e r a t i o n o f a n t i b o d y r e s p o n s e s . However, t h e a d d i t i o n o f 2 m e r c a p t o e t h a n o l , w h i c h was i n c l u d e d i n t h e c u l t u r e s , has been shown t o e l i m i n a t e t h e need f o r macrophages i n v i t r o (Chen, 1972) . The i n h i b i t o r y e f f e c t s o f GVH serum i s n o t H-2 s p e c i f i c because i t caused a 93% i n h i b i t i o n o f t h e 2 way MLC i n w h i c h one o f t h e r e s p o n d i n g c e l l s  25  was a l l o g e n e i c t o t h e s o u r c e o f serum.  Only a 50% i n h i b i t i o n o f the MLC would  have o c c u r r e d i f t h e serum i n h i b i t o r was H-2 s p e c i f i c . r e s p o n s i b l e f o r t h e immunosuppression  The serum f a c t o r  does n o t appear t o be a l l o a n t i b o d y ,  w h i c h i s sometimes p r e s e n t i n t h e serum o f GVH mice (Lapp, W.S., P e r s o n a l Communication), because t h e GVH serum i n h i b i t e d t h e Con A r e s p o n s e o f normal DBA/2J c e l l s , which a r e o f t h e same genotype as t h e c e l l s used t o i n d u c e t h e GVH. The i n h i b i t o r y m a t e r i a l i n t h e serum o f GVH mice e l u t e d on a B i o - G e l P-200 column w i t h t h e immunoglobulin f r a c t i o n o f serum.  This i n d i c a t e s that the  i n h i b i t o r y m a t e r i a l has a m o l e c u l a r w e i g h t o f 150,000 o r g r e a t e r .  Although  immunoglobulin i s t h e main component o f t h i s f r a c t i o n t h e r e a r e s e v e r a l o t h e r p r o t e i n s w i t h s i m i l a r m o l e c u l a r w e i g h t s such as t h e complement p r o t e i n s . F u r t h e r p u r i f i c a t i o n o f t h e i n h i b i t o r y m a t e r i a l i s needed b e f o r e one can c o n c l u d e any a d d i t i o n a l i n f o r m a t i o n o n . i t ' s c h e m i c a l n a t u r e and s o u r c e . I t i s p l a u s a b l e t o assume t h a t i n h i b i t o r y m a t e r i a l i n t h e serum o f GVH mice i s r e s p o n s i b l e f o r t h e g e n e r a l i z e d immunosuppression w h i c h GVHD.  accompanies  A s i m i l a r f a c t o r ( s ) , o b t a i n e d from c e l l f r e e s u p e r n a n t s o f c u l t u r e d  s p l e e n c e l l s from GVH mice, has been r e c e n t l y r e p o r t e d which s u p p r e s s e s t h e PHA response o f normal s p l e e n c e l l s ( P h i l l i p s , 1975).  T h i s f a c t o r ( s ) might be  an immunoregulatory m o l e c u l e produced by s u p p r e s s o r c e l l s o r i t may a c t i v a t e s u p p r e s s o r c e l l s w h i c h i n t u r n s u p p r e s s t h e f u n c t i o n o f o t h e r lymphoid c e l l s . I t has a l s o been found t h a t t h e serum o f tumour b e a r i n g a n i m a l s a l s o c o n t a i n s a f a c t o r w h i c h s i m i l a r l y e l u t e s w i t h immunoglobulin and s u p p r e s s e s the  f u n c t i o n o f normal lymphoid c e l l s ( L e v y , 1975) .  These i m m u n o r e g u l a t i n g  m o l e c u l e s may be produced by a h o s t i n response t o an abnormal a n t i g e n i c l o a d such as tumour o r i n t h e case o f an a n i m a l u n d e r g o i n g a GVHR be produced by the  grafted c e l l s which a r e i n constant contact w i t h f o r e i g n antigen.  26  BIBLIOGRAPHY  1.  B i n z , H., W i g z e l l , H. 1975. Shared i d i o t y p i c d e t e r m i n a n t s on B and T lymphocytes r e a c t i v e a g a i n s t t h e same a n t i g e n i c d e t e r m i n a n t s . J . Exp. Med. 142:197.  2.  C a n t o r , H., A s o f s k y , R. 1970. Synergy among lymphoid c e l l s m e d i a t i n g t h e GVH response. J . Exp. Med. 131 :215.  3.  Chen, C., H i r s c h , J.G. 1972. The e f f e c t s o f m e r c a p t o e t h a n o l and o f p e r i t o n e a l macrophages on t h e a n t i b o d y - f o r m i n g c a p a c i t y o f nonadherent mouse s p l e e n c e l l s i n v i t r o . J . Exp. Med. 136:604.  4.  Claman, H.N., Chaperon, E.A., Hayes, L . L . 1969. Thymus-marrow immunocompetence. IV. The growth and immunocompetence o f t r a n s f e r r e d marrow, thymus, and s p l e e n c e l l s i n p a r e n t and F i h y b r i d m i c e . T r a n s p l a n t a t i o n 7_:87.  5.  Cunningham, A . J . , S z e n b e r g , A. 1968. F u r t h e r improvements i n t h e p l a q u e technique f o r d e t e c t i n g s i n g l e antibody-forming c e l l s . Immunology. 14/. 599.  6.  D a v i s , W.E., C o l e , L . J . , S c h a f f e r , W.T. 1970. GVHR i n n o n - i r r a d i a t e d mice. E a r l y s u p p r e s s i o n o f J e r n e p l a q u e s and h e m o p o i e t i c c o l o n y f o r m i n g u n i t s . T r a n s p l a n t a t i o n . 9_:529.  7.  D e c k e r s , J . , D a v i e s , R.C., P a r k e r , G.A. 1973. The e f f e c t o f tumour s i z e on concommitant tumour immunity. Cancer Res. 33 :33.  8.  E l i e , R., Lapp, W.S. 1975. GVH immunosuppression: r e g u l a t o r y f u n c t i o n o f macrophages i n t h e humoral immune response i n Immune R e c o g n i t i o n , Academic P r e s s , I n c .  9.  E l k i n s , W.L. 1966. I n t e r a c t i o n o f donor and h o s t lymphoid c e l l s i n t h e p a t h o g e n e s i s o f r e n a l c o r t i c a l d e s t r u c t i o n induced by a l o c a l GVHR. J . Exp. Med. 123:103.  10.  E l k i n s , W.L. 1971. C e l l u l a r immunology and t h e p a t h o g e n e s i s o f GVHR. Prog. A l l e r g y . 15:81.  11.  F i e l d , E.O., G i w s , J.E. 1966. Reduced s e n s i t i v i t y o f F i h y b r i d r a t s t o re-challenge w i t h parental s t r a i n spleen c e l l s . C l i n . Exp. Immunol. 1_:195.  12.  F i e l d , E.O., L a u c h i , M.N., G i b b s , J.E. 1967. The t r a n s f e r o f r e f r a c t o r i n e s s t o GVHD i n 7i h y b r i d r a t s . T r a n s p l a n t a t i o n . 5_:241.  13.  G r a f , M.W., U h r , J.W. 1969. R e g u l a t i o n o f a n t i b o d y f o r m a t i o n b y serum a n t i b o d y . J . Exp. Med. 130:1175.  14.  F r u s h k a , M., Lapp, W.S. 1974. E f f e c t o f lymphoid t i s s u e t r a n s p l a n t s on the i n t e n s i t y o f an e x i s t i n g GVHR. T r a n s p l a n t a t i o n . 17 :157 .  15.  Haran-Ghera, N. , Ben-Yaakov, A., P e l e d , A. , B e n t w i c k , Z. 1973. Immune s t a t u s o f S J L / J mice i n r e l a t i o n t o age and spontaneous tumour development. J . N a t l . Cancer I n s t . 50:1227.  27  16.  J a c o b s , D.M., M o r r i s o n , D.C.J. 1975. S t i m u l a t i o n o f a T-independent p r i m a r y a n t i - h a p t e n response i n v i t r o by T N P - l i p o p o l y s a c c h a r i d e (TNP-LPS). J . Immunology. 114:360.  17.  K a t z , D.H., B e n c e r r a f , B. The r e g u l a t o r y i n f l u e n c e o f a c t i v a t e d T c e l l s on B c e l l r e s p o n s e s t o a n t i g e n . Adv. Immunol. 15:1.  18.  Lapp, W.S., M o l l e r , G. 1969. P r o l o n g e d s u r v i v a l o f H-2 i n c o m p a t i b l e s k i n g r a f t s o f F i a n i m a l s t r e a t e d w i t h p a r e n t a l lymphoid c e l l s . Immunology. 17_: 339 .  19.  L e w i s , R.M., Armstrong, M.V.K., Andre-Schwartz, J . , M u f t v o f h v , A., B e l d o t t i , L., Schwartz, R.S. 1968. C h r o n i c a l l o g e n i c d i s e a s e . I . Development o f g l o m e r u l o n e p h r i t i s . J . Exp. Med. 128 :653.  20.  L e v y , J.G., Smith, A.G., Whitney, R.B., McMaster, R., K i l b u r n , D.G. 1976. C h a r a c t e r i z a t i o n o f a T-lymphocyte i n h i b i t o r i n t h e serum o f tumour b e a r i n g mice. Immunology, i n p r e s s .  21.  L o n a i , P., M o g i l n e r , B., R o t t e r , V., T r a i n i n , H. 1973. S t u d i e s on t h e e f f e c t o f a thymic f a c t o r on d i f f e r e n t i a t i o n o f thymus-derived lymphocytes. E u r . J . Immunol. 3_:21.  22.  M i l l e r , J.A.F.P., M i t c h e l l , G.F., W e i s s , G.P. 1967. C e l l u l a r b a s i s o f the i m m u n o l o g i c a l d e f e c t s i n thymectomized mice. N a t u r e . 214 :992.  23.  M o l l e r , G. 1971. S u p p r e s s i v e e f f e c t o f GVHR on t h e immune response t o heterologous red c e l l s . Immunology. 20 :597 .  24.  P h i l l i p s , S.M., Gleichmann, H., H i r s c h , M.S., B l o c k , P., M e r r i l , J . P . , Schwartz, R.S., C a r p e n t e r , C B . 1975. C e l l u l a r immunity i n t h e mouse. V. F u r t h e r s t u d i e s on l e u k e m i a v i r u s a c t i v a t i o n i n a l l o g e n e i c r e a c t i o n s o f mice s t i m u l a t o r y parameters. C e l l . Immunol. 15 :169.  25.  Pope, B.L., Whitney, R.B., L e v y , J.G. 1976. Suppressor c e l l s i n t h e s p l e e n s o f tumour b e a r i n g mice. J . Immunology, i n p r e s s .  26.  Rowely, D.A., F i t c h , F.W., S t u a r t , F.P., K o h l e r , H., C o s e y z a , H. 1973. S p e c i f i c s u p p r e s s i o n o f immune r e s p o n s e s . S c i e n c e . 181:1133.  27.  S c o l l a y , R.G., Hoffman, F., G l o b e r s o n , A. 1974. GVHR i n F i r e c i p i e n t s i n t h e absence o f donor ( p a r e n t a l ) c e l l p r o l i f e r a t i o n . E u r . J . Immunol. 4_:490.  28.  Simonsen, M. 1957. GVHR, t h e i r n a t u r a l h i s t o r y and a p p l i c a b i l i t y as t o o l s i n r e s e a r c h . A c t a . P a t h o l . M i c r o b i o l . Scand. 40:480.  29.  Smith, R.T., Kondu, S.- 1973. The s t i m u l a t o r y e f f e c t s o f b e a r i n g p r i m a r y m e t h y l c h o l a n t h r e n e - i n d u c e d tumours upon t h e murine l y m p h o r e t i c u l a r system. I n t . J . Cancer. 12:577.  30.  S t r a u s b a c h , P.A., S u l i c a , A., G i v a l , D. 1970. G e n e r a l method f o r t h e d e t e c t i o n o f c e l l s p r o d u c i n g a n t i b o d y a g a i n s t haptens and p r o t e i n s . N a t u r e . 227:68.  31.  T a y l o r , R.B. 1963. Immunological competence o f thymus c e l l s t r a n s f e r t o thymetomized a n i m a l s . N a t u r e . 199:873.  after  28  32.  Thomas, E.D., S t o r b , R. , E p s t e i n , R.B. 1969. Symposium on bone marrow transplantation. T r a n s p l a n t . P r o c . 1_:31.  33.  Whitney, R.B., L e v y , J.G., S m i t h , A.G. 1974. I n f l u e n c e of tumour s i z e and s u r g i c a l r e s e c t i o n on c e l l - m e d i a t e d .immunity i n mice. J . N a t l . Cancer I n s t . 53:111.  34.  Whitney, R.B., L e v y , J.G. 1975. E f f e c t s of s e r a from tumour b e a r i n g mice on mitogen and a l l o g e n e i c c e l l s t i m u l a t i o n of normal lymphoid c e l l s . J . N a t l . Cancer I n s t . 54:733.  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0100124/manifest

Comment

Related Items