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UBC Theses and Dissertations

The effect of the W gene on immune responses in mice McNay, Margaret Lynne 1975

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THE  EFFECT OF THE W GENE ON  IMMUNE RESPONSES IN MICE  by  MARGARET LYNNE McNAY B.Ed., University of B r i t i s h Columbia, 1969  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE  REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Zoology  We accept this thesis as conforming to the require^ standard  THE  UNIVERSITY OF BRITISH COLUMBIA September, 1975  In p r e s e n t i n g t h i s  thesis  an advanced degree at the L i b r a r y s h a l l I  fulfilment of  the U n i v e r s i t y of B r i t i s h  make i t  freely available  f u r t h e r agree t h a t p e r m i s s i o n  for  for  the requirements f o r  Columbia,  I agree  r e f e r e n c e and  f o r e x t e n s i v e copying o f  this  that  study. thesis  s c h o l a r l y purposes may be granted by the Head o f my Department or  by h i s of  in p a r t i a l  this  written  representatives. thesis  is understood that  f o r f i n a n c i a l gain s h a l l  permission.  Department of  ~~**?f ^ f^~ n  J:rc  The U n i v e r s i t y of B r i t i s h 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  Date  It  L SkJ-*l*^  Columbia  copying o r p u b l i c a t i o n  not be allowed without my  ABSTRACT  P r e v i o u s r e p o r t s t h a t lymphocytes from W/W  v  mice m i g r a t e more  s l o w l y than do lymphocytes from normal mice, and t h a t s p l e e n s from W/W  mice produce fewer p l a q u e - f o r m i n g c e l l s  V  z a t i o n w i t h sheep r e d b l o o d c e l l s  i n response t o immuni-  (SRBC) than do normal mice, s u g g e s t e d  t h a t o t h e r immune responses i n t h e s e mice might a l s o be a f f e c t e d . The f o l l o w i n g experiments have r e v e a l e d t h a t g r a f t is  f a s t e r and more v i g o u r o u s among W/W  V  rejection  mice than among +/+,  normal  mice: 1) s t r o n g l y a n t i g e n i c , H-2 i n c o m p a t i b l e , s k i n a l l o g r a f t s examined logically  a t the e i g h t h day a f t e r t r a n s p l a n t a t i o n t o W/W  V  hosts exhibit  h i g h e r g r a f t r e j e c t i o n s c o r e s than do i d e n t i c a l s k i n a l l o g r a f t s p l a n t e d t o +/+  histo-  trans-  hosts;  2) weakly a n t i g e n i c s k i n a l l o g r a f t s b e a r i n g the H-Y a n t i g e n , when t r a n s p l a n t e d t o female +/+ and W/W  v  W/W  V  h o s t s than by +/+  h o s t s , a r e r e j e c t e d more q u i c k l y by  hosts;  3) the uptake o f t r i t i a t e d thymidine by c e l l s of mice b e a r i n g s k i n a l l o g r a f t s  i n the p e r i p h e r a l b l o o d  i n d i c a t e s a g r e a t e r p r o p o r t i o n of a c t i -  v a t e d c e l l s i n the c i r c u l a t i o n o f W/W  V  mice than o f +/+  mice.  C o n f i r m a t i o n has a l s o been o b t a i n e d t h a t some a n t i b o d y responses among W/W  V  W/W  V  mice a r e d e p r e s s e d .  I n response to immunization w i t h SRBC,  mice produce about 1/4 the number o f p l a q u e - f o r m i n g c e l l s as do  +/+ mice.  W/+  and W /+ V  mice produce i n t e r m e d i a t e numbers o f p l a q u e ii  forming c e l l s . a secondary +/+  T i t e r s o f SRBC a g g l u t i n i n s produced by W/W  response are s i g n i f i c a n t l y  lower than t i t e r s produced  by  mice. The  uptake o f t r i t i a t e d  v/ith the B - c e l l mitogen in  mice i n  LPS  thymidine by s p l e e n c e l l s  indicates  t h e i r response t o t h i s mitogen.  that W/W  V  Treatment  cells  are  stimulated deficient:  w i t h the T - c e l l  mitogen  Concanavalin-A, however, r e s u l t s i n m a r g i n a l l y g r e a t e r thymidine by W/W  V  spleen c e l l s  than by +/+  T - c e l l responses g e n e r a l l y may  cells.  These  r e s u l t s suggest  be somewhat enhanced and B - c e l l  uptake  that responses  v d e p r e s s e d i n mice o f W/W  genotype.  The a l t e r e d r e a c t i v i t y of lymphocytes  from W/W  V  mice can be  accounted f o r by p o s t u l a t i n g an a l t e r a t i o n i n the s u r f a c e t i c s o f these c e l l s .  characteris-  The h y p o t h e s i s t h a t the W l o c u s c o n t r o l s a  cell  s u r f a c e c h a r a c t e r i s t i c i s the o n l y h y p o t h e s i s y e t f o r m u l a t e d which is  a b l e to account f o r a l l the better-known  the W m u t a t i o n  (anemia, s t e r i l i t y ,  as f o r i t s i m m u n o l o g i c a l e f f e c t s .  pleiotropic effects  of  and a l a c k o f p i g m e n t a t i o n ) as w e l l T h i s h y p o t h e s i s and i t s i m p l i c a t i o n s  are d i s c u s s e d i n d e t a i l i n the t e x t .  TABLE OF CONTENTS  Page ABSTRACT  .  • • •  1  1  LIST OF TABLES  ix  LIST OF FIGURES  xi  LIST OF ABBREVIATIONS ACKNOWLEDGEMENT  x i i  .  xiv  INTRODUCTION  • • •  1  MATERIALS AND METHODS  11  I. MICE  11  I I . SKIN GRAFT STUDIES  .  A. T r a n s p l a n t a t i o n  Technique  12  B. Measurement o f A l l o g r a f t S u r v i v a l C. E s t a b l i s h m e n t Allograft  11  13  o f C r i t e r i a by Which t o Measure  Survival  16  1) I s o g r a f t S t u d i e s  16  2) A l l o g r a f t S t u d i e s  17  a) D e t e r m i n a t i o n o f h i s t o l o g i c a l c r i t e r i a f o r assessing a l l o g r a f t s u r v i v a l b) D e t e r m i n a t i o n o f MST o f DBA/2 g r a f t s on C57B1/6 h o s t s D. A l l o g r a f t R e j e c t i o n i n W/W A c r o s s H-2 B a r r i e r s  Mice: G r a f t s Exchanged  E. A l l o g r a f t R e j e c t i o n i n W/W  Mice: G r a f t s Exchanged  V  V  iv  18  18  A c r o s s Weak H i s t o c o m p a t i b i l i t y B a r r i e r s F. H i s t o l o g i c a l P r e p a r a t i o n  17  of Skin Grafts  20 21  V  Page I I I . LYMPHOCYTE ACTIVATION STUDIES  . . . .  21  A. L a b e l l i n g iri v i v o  22  B. L a b e l l i n g i n v i t r o  22  C. S c i n t i l l a t i o n  Counting  . . .  IV. HUMORAL IMMUNE RESPONSES  24  A. P r o d u c t i o n o f SRBC A g g l u t i n i n s i n +/+ B. P r o d u c t i o n and W/W  v  23  of Plaque-forming  and W/W  C e l l s i n +/+,  V  W/+,  Mice .  25  W /+, V  Mice  26  V. MITOGEN STIMULATION STUDIES  28  VI. OTHER STUDIES  . . . .  31  A. Macrophage M i g r a t i o n S t u d i e s  31  B. Lymphocyte Adhesion S t u d i e s  32  V I I . HAEMATOLOGICAL TECHNIQUES  36  RESULTS  37  I . SKIN GRAFT STUDIES  37  A. H i s t o l o g i c a l F e a t u r e s  of Skin I s o g r a f t Healing  B. H i s t o l o g i c a l F e a t u r e s  of Skin A l l o g r a f t Rejection . .  1) G e n e r a l  . . .  Observations  37 41 41  a) The h e a l i n g - i n phase  41  b) The a c u t e  42  r e j e c t i o n phase  c) The replacement phase 2) H i s t o l o g i c a l C r i t e r i a  52  f o r A s s e s s i n g Degree o f  Allograft Survival  52  3) MST o f DBA/2 S k i n A l l o g r a f t s on C57B1/6 +/+ Mice C. A l l o g r a f t R e j e c t i o n i n W/W  V  Mice  1) On the E i g h t h Day A f t e r T r a n s p l a n t a t i o n  60 60  . . . .  60  vi  Page 2) MST of DBA S k i n A l l o g r a f t s on +/+, W/WV Hosts  W/+,  W /+, V  60  3) G r a f t s Exchanged A c r o s s Weak H i s t o c o m p a t i b i l i t y Barriers I I . LYMPHOCYTE ACTIVATION STUDIES  .64 66  A. L a b e l l i n g i n v i v o  66  B. L a b e l l i n g i n v i t r o  66  I I I . HUMORAL IMMUNE RESPONSES  70  A. P r o d u c t i o n of SRBC A g g l u t i n i n s i n +/+ B. P r o d u c t i o n of Plaque-forming and W/W  v  and W/W  C e l l s i n +/+,  V  W/+,  Mice . W /+, V  Mice  73  IV. MITOGEN STIMULATION STUDIES  75  V. OTHER STUDIES VI.  70  78  A. Macrophage M i g r a t i o n S t u d i e s  78  B. Lymphocyte Adhesion  79  Studies  V I . HAEMATOLOGY  .  A. U n g r a f t e d Mice B. Mice B e a r i n g S k i n A u t o g r a f t s o r S k i n A l l o g r a f t s DISCUSSION  80 80  . . .  81 83  I . TRANSPLANTATION IMMUNITY A.  Genetics of T r a n s p l a n t a t i o n  83 83  1) C h a r a c t e r i s t i c s of T r a n s p l a n t e d T i s s u e  83  2) H i s t o c o m p a t i b i l i t y Genes  86  3) Other Genes A f f e c t i n g Immune Responses  87  B. R e c o g n i t i o n and S e n s i t i z a t i o n  88  1) A n t i g e n R e c o g n i t i o n by Lymphocytes  88  2) S e n s i t i z a t i o n  89  vii  Page C. The  R e j e c t i o n Process  94  1) Role of Lymphocytes  94  2) The  95  Cellular Infiltrate  3) E f f e c t o r Mechanisms i n A l l o g r a f t R e j e c t i o n a) S p e c i f i c i t y o f g r a f t b)  D.  Contact-induced  . . .  rejection  98 98  cell-mediated cytotoxicity  .  99  c) Role of lymphokines  100  d) Role of antibody  102  e) C o n c l u s i o n  103  S k i n A l l o g r a f t R e j e c t i o n i n W/W  V  1) MST  o f F i r s t - s e t H-2  Mice  104  Incompatible  Grafts  2) G r a f t R e j e c t i o n at the E i g h t h Day plantation  After  . . . .  104  Trans104  3) R e j e c t i o n of S k i n A l l o g r a f t s A c r o s s Weak HBarriers  106  4) A Study of the C e l l u l a r I n f i l t r a t e  ( C o l l e n , 19 74)  I I . LYMPHOCYTE ACTIVATION STUDIES  106 107  A.  General  Discussion  107  B.  Lymphocyte A c t i v a t i o n S t u d i e s  i n +/+  and W/W  V  Mice. .  110  C. A C o r r e l a t i o n of O b s e r v a t i o n s from Lymphocyte A c t i v a t i o n S t u d i e s and A l l o g r a f t R e j e c t i o n S t u d i e s i n +/+  Mice  110  I I I . HUMORAL IMMUNE RESPONSES  114  A.  and W/W  General  v  Discussion  114 v  B. Humoral Immune Responses i n +/+  and W/W  Mice . . . .  118  ...  120  IV. MITOGEN STIMULATION STUDIES A.  General  Discussion  B. Mitogen S t i m u l a t i o n S t u d i e s  120 i n +/+  and  W/W  V  Mice  . .  125  viii  Page V. OTHER STUDIES  127  A. Macrophage M i g r a t i o n B. Lymphocyte Adhesion  127 . . •  127  V I . HAEMATOLOGY V I I . SPECULATION: THE PRIMARY SITE OF ACTION OF THE W GENE  129 . .  130  CONCLUSION  138  REFERENCES  139  LIST OF  TABLES  Table I. II.  III.  IV.  Page Histological  features of s k i n  isograft healing  H i s t o l o g i c a l f e a t u r e s of s i x s t a g e s of r e j e c t i o n i n normal mice  VII.  VIII.  IX.  X.  XI.  allograft 58  D i s t r i b u t i o n o f g r a f t r e j e c t i o n s c o r e s of DBA/2 s k i n a l l o g r a f t s on +/+, W/+, W /+, and W/W mice; g r a f t s were e x c i s e d on the e i g h t h day a f t e r t r a n s p l a n t a t i o n . Mortality W /+, and v  VI.  38  G r a f t r e j e c t i o n s c o r e s determined by h i s t o l o g i c a l a n a l y s i s of DBA/2 s k i n a l l o g r a f t s on +/+ h o s t s . . . .  v  V.  skin  . . . .  61  v  d a t a f o r DBA/2 s k i n a l l o g r a f t s on +/+, W/W hosts v  .  62  W/+, /. .  63  G r a f t r e j e c t i o n s c o r e s f o r g r a f t s of s k i n from +/+ male donors t r a n s p l a n t e d to +/+ and W/WV female h o s t s  .  65  Uptake of t r i t i a t e d thymidine by c e l l s o f the p e r i p h e r a l b l o o d o f mice b e a r i n g s k i n a l l o g r a f t s . Labelling done i n v i v o  67  Uptake of t r i t i a t e d thymidine by c e l l s of the p e r i p h e r a l b l o o d of mice b e a r i n g s k i n a l l o g r a f t s . Labelling done ixi v i t r o  68  Serum a g g l u t i n i n t i t e r s i n mice of a f t e r immunization w i t h SRBC  71  different  genotype  A.  Numbers of plaque-forming c e l l s produced by s p l e e n s of mice of d i f f e r e n t genotype f o u r days a f t e r exposure t o SRBC B. Numbers of plaque-forming c e l l s produced by s p l e e n s of mice of d i f f e r e n t ^ g e n o t y p e s i x days a f t e r exposure to SRBC  Uptake of t r i t i a t e d thymidine ( e x p r e s s e d as mean CPM) by s p l e e n c e l l s of +/+ and W/W mice c u l t u r e d w i t h v a r i o u s c o n c e n t r a t i o n s of LPS and Con-A  74  74  v  76  v XII.  S t i m u l a t i o n i n d i c e s of s p l e e n c e l l s from +/+ and mice c u l t u r e d w i t h v a r i o u s c o n c e n t r a t i o n s of LPS Con-A and l a b e l l e d w i t h t r i t i a t e d thymidine ix  W/W and 77  X  Table XIII.  XIV.  XV.  Page A r e a (mm ) c o v e r e d by p e r i t o n e a l c e l l s from mice o f d i f f e r e n t genotype a f t e r 8 hours o f m i g r a t i o n from c a p i l l a r y tubes i n Mackaness-type chambers  79  P e r c e n t a g e o f lymph node c e l l s a d h e r i n g t o g l a s s cover s l i p s a f t e r 45 minutes i n c u b a t i o n  80  T o t a l and d i f f e r e n t i a l w h i t e b l o o d c e l l u n g r a f t e d +/+, W/+, W /+, and W/W mice  81  2  v  XVI.  T o t a l and d i f f e r e n t i a l w h i t e b l o o d c e l l counts o f +/+, W/+, W /+, and W/W mice b e a r i n g s k i n a u t o g r a f t s o r skin allografts  82  A summary o f major experiments c o n c e r n i n g immune responses i n W mutant mice  84  v  XVII.  counts o f  v  v  LIST OF FIGURES  Figure 1.  Page a. b.  Normal mouse skin Relationship between graft and host t i s s u e . . . .  14  2.  Construction of PFC plates  29  3.  Mackaness-type chamber for c e l l migration studies. . .  33  4.  Ring chambers for c e l l adhesion studies  35  5.  Skin i s o g r a f t s  39  6.  Skin a l l o g r a f t s i n the h e a l i n g - i n phase  43  7.  Skin a l l o g r a f t s entering the acute r e j e c t i o n phase . .  45  8.  Skin a l l o g r a f t s undergoing acute r e j e c t i o n  47  9.  Skin a l l o g r a f t s undergoing acute r e j e c t i o n  47  10.  -  Skin a l l o g r a f t s i n the l a t e r part of the acute r e j e c t i o n phase  50  11.  Skin a l l o g r a f t s entering the replacement phase . . . .  50  12.  Skin a l l o g r a f t s i n the replacement phase  53  13. 14.  Skin a l l o g r a f t s i n the replacement phase Uptake of t r i t i a t e d thymidine by c e l l s of the peripheral blood of mice bearing skin a l l o g r a f t s or skin autografts. L a b e l l i n g done i n v i t r o  55  69  Serum agglutunin t i t e r s of mice of d i f f e r e n t genotype a f t e r immunization with SRBC  72  A s i m p l i f i e d and schematic representation of some of the events occurring during the course'of a c e l l u l a r immune response  90  15.  16.  xi  LIST OF ABBREVIATIONS  AFC  antibody-forming  cell  ARC  antigen-reactive  cell  B-cell  thymus-independent  BSA  b o v i n e serum albumen  BSS  balanced s a l t  cAMP  c y c l i c adenosine monophosphate  Ci/mM  Curies per millimole  Con-A  Concanavalin-A  CPM  counts p e r minute  DPM  disintegrations  ECF  eosinophil  H  histocompatibility  IgG  immunoglobulin  G  IgM  immunoglobulin  M  lr  immune  LAF  lymphocyte  LPS  lipopolysaccharide  LT  lymphotoxin  MIF  macrophage i n h i b i t i o n  MST  median s u r v i v a l  PFC  plaque-forming  PHA  phy t o h e m a g g l u t i n i n  POPOP  1,4-Bis-[2-(5-phenyloxazolyi)]-benzene  cell  solution  p e r minute  chemotactic f a c t o r  response activation  factor  factor  time cell  xii  xiii  PPO  2,5-diphenyloxazole  PWM  pokeweed mitogen  RPMI  Roswell  S.A.  specific activity  SRBC  sheep red blood c e l l s  TCA  t r i c h l o r o a c e t i c acid  T-cell  thymus-dependent  TF  transfer  Park Memorial I n s t i t u t e (tissue culture medium)  factor  cell  ACKNOWLEDGEMENT  I should l i k e o r i g i n a l problem  to thank Dr. A. B. Acton  f o r s u g g e s t i n g the  from which t h i s t h e s i s has d e v e l o p e d ,  and f o r h i s  a d v i c e and encouragement d u r i n g the c o u r s e o f t h i s study.  I also  wish  t o thank the members o f my T h e s i s Committee, Dr, C. V. Finnegan, Dr. H. K. K a s i n s k y , and Dr. J . W. Thomas, f o r t h e i r  comments and c r i t i c i s m s  r e g a r d i n g my r e s e a r c h and the p r e p a r a t i o n o f t h i s  thesis.  I am v e r y g r a t e f u l t o M. Douglas f o r e x c e l l e n t t e c h n i c a l  assis-  tance a t s e v e r a l s t a g e s i n t h i s work, and f o r the d r a f t i n g o f t h e f i g u r e s in this thesis.  In a d d i t i o n , I s h o u l d l i k e  t o thank R. McMaster o f  the Department o f M i c r o b i o l o g y f o r h i s h e l p w i t h t h e t e c h n i q u e s i n the mitogen s t i m u l a t i o n experiments,  used  and Dr. J . Levy, a l s o o f the  Department o f M i c r o b i o l o g y , f o r the use o f c e r t a i n m a t e r i a l s and f a c i l i ties i n her laboratory. F i n a l l y , I wish  t o say a s p e c i a l  thank y o u t o my c o l l e a g u e s ,  i n p a r t i c u l a r P a t C o l l e n , C. Boogaard, and E. S l a v i n s k i , w i t h whom I have s h a r e d many hours  o f d i s c u s s i o n and who have been a c o n t i n u a l  s o u r c e o f encouragement t o me. D u r i n g the course o f t h i s s t u d y , I have been s u p p o r t e d by a b u r s a r y and a s c h o l a r s h i p from the N a t i o n a l Research  xiv  C o u n c i l o f Canada.  INTRODUCTION  A mutation  a t the W l o c u s i n mice was  first  r e c o g n i z e d by i t s  e f f e c t on coat c o l o u r and pigment p a t t e r n (Durham, 1908). however, when homozygous anemia and  The  mutation,  i s also r e s p o n s i b l e f o r a severe macrocytic  for s t e r i l i t y .  Mice demonstrating  these three  u n r e l a t e d e f f e c t s have proven p a r t i c u l a r l y s u i t a b l e s t u d i e s i n c e l l and developmental  biology:  seemingly  for a variety  experiments  of  involving  pro-  s p e c t i v e pigment c e l l s have l e d to a g r e a t e r u n d e r s t a n d i n g o f the g e n e t i c c o n t r o l o f s p o t t i n g p a t t e r n s ; s t u d i e s of the anemia have y i e l d e d mation  r e l a t i n g t o the p r o c e s s e s and c o n t r o l o f h e m a t o p o i e s i s ;  i n v e s t i g a t i o n s o f the s t e r i l i t y  taken the i n v e s t i g a t i o n s  and immune r e s p o n s e s .  described i n this  s t u d i e s as i t has  In the p r e s e n c e W mutation  I have under-  t h e s i s i n the hope o f demon-  i n developmental  on p i g m e n t a t i o n  i s dominant, and h i s t o r i c a l l y (Gruneberg,  i s p r e s e n t w i t h m o d i f y i n g genes which cause dominant s p o t t i n g gene. , W  studies.  o f c e r t a i n m o d i f y i n g genes, the e f f e c t o f the  been c a l l e d "dominant s p o t t i n g "  , W  certain  t h a t the W mutant might prove as i n t e r e s t i n g and u s e f u l i n  immunological  W  In a d d i t i o n ,  t h a t the e f f e c t s o f t h i s p l e i o t r o p i c gene might  i n c l u d e an e f f e c t on lymphocytes  strating  and  o f these mice have h e l p e d to e s t a b l i s h  the e x t r a - g o n a d a l o r i g i n o f p r i m o r d i a l germ c e l l s . o b s e r v a t i o n s suggest  infor-  1952).  t h i s gene has  More commonly, W  i t to behave as a semi-  A number of a l l e l e s e x i s t  at the W l o c u s ,  , W , and W , each a f f e c t i n g p i g m e n t a t i o n p a t t e r n s i n  W,  2  some way  (Green, 1966).  Mice carrying any one of these a l l e l e s exhibit  variable white spotting and sometimes a s l i g h t d i l u t i o n of colour as well.  Mice carrying any two W a l l e l e s are, except for t h e i r eyes and  occasionally the tips of t h e i r ears, e n t i r e l y white.  Such mice are  also s t e r i l e and exhibit a severe anemia which i s usually l e t h a l within a few days of b i r t h .  Any W a l l e l e i n combination with the W  V  allele,  however, produces a less severe anemia and the animal survives. of the W locus have, therefore, u t i l i z e d either W/W or W/W V  V  V  Studies  mice almost  exclusively. Numerous genes a f f e c t i n g pigmentation have been i d e n t i f i e d i n mice, but those responsible f o r the various white spotting phenotypes have p a r t i c u l a r l y interested developmental b i o l o g i s t s .  Spotting i s  e t i o l o g i c a l l y d i f f e r e n t from such colour anomalies as albinism.  In  albino animals, melanocytes are d i s t r i b u t e d normally i n the tissues and are present i n normal numbers, but are genetically incapable of producing tyrosinase, and hence can not produce pigment.  In contrast,  spotting genes result i n a complete absence of melanocytes from the spotted area ( S i l v e r s , 1956). The spotting genes have been shown to a f f e c t pigment c e l l development v i a two routes, either d i r e c t l y , v i a the neural crest from which the pigment c e l l s a r i s e , or i n d i r e c t l y , v i a the environment through which the pigment c e l l s must t r a v e l or i n which they d i f f e r e n t i a t e (Markert and S i l v e r s , 1956).  Belted (bt) and S t e e l (SI), f o r example,  cause certain tissue environments to be unsuitable for the d i f f e r e n t i a t i o n or s u r v i v a l of melanoblasts of any genotype (Mayer and Maltby,  3 1964;  Mayer and Green, 1968).  Dominant spotting (W), on the other  hand, acts v i a the neural crest: by this gene precludes  some developmental defect caused  the production or d i f f e r e n t i a t i o n of tnelano-  blasts (Mayer and Green, 1968).  Experiments suggest that i n W embryos  melanoblasts may be produced i n smaller numbers than normal, and with a decreased a b i l i t y to survive or d i f f e r e n t i a t e (Mayer, 1970). v The s t e r i l i t y of W/W  mice i s associated with a profound defect  i n the p r o l i f e r a t i o n of primordial germ c e l l s (Mintz and R u s s e l l , 1957). In normal embryos, primordial germ c e l l s appear i n the yolk sac e p i thelium on the eighth day of development, and during the next four days migrate along the dorsal gut mesentery towards the g e n i t a l ridges, p r o l i f e r a t i n g markedly as they t r a v e l .  In W/W  V  embryos, primordial  germ c e l l s are present i n normal numbers at eight days, but p r o l i f e r a t e very l i t t l e a f t e r t h i s .  The c e l l s are also retarded i n migration,  so that very few c e l l s ever reach the g e n i t a l ridges. newborn W/W  V  The gonads of  mice are thus severely d e f i c i e n t i n germ c e l l s , and the  adult animals are s t e r i l e . Of a l l hereditary mouse anemias, the severe macrocytic exhibited by W/W  V  1966).  disorder  mice has been the most extensively studied (Green,  The W series anemia i s characterized by a reduction i n erythro-  cyte numbers and an increase i n mean c e l l volume.  The s e v e r i t y of the  disease depends on which a l l e l e s of the gene are present:  some com-  binations are l e t h a l , causing general anoxemia i n a l l tissues and r e s u l t i n g i n death i n utero or within a few days of b i r t h (Borghese, 1959).  4 v  The anemia of W/W  mice can be permanently cured by the trans-  plantation of marrow from normal, coisogenic animals (Bernstein and Russell, 1959); the transplanted c e l l s apparently c e l l population and quickly repopulate  outgrow the indigenous  the anemic host.  The  defect  produced by the W gene, therefore, c l e a r l y resides within the mutant hematopoietic c e l l s themselves, and i s not mediated by humoral or t o x i c factors i n the environment, or by the absence of normal hematopoietic stimuli.  v Marrow from W/W genic +/+  mice, when transplanted to i r r a d i a t e d , coiso-  r e c i p i e n t s , i s severely d e f i c i e n t i n i t s capacity to form  macroscopic spleen colonies (McCulloch,  Siminovitch, and T i l l , 1964).  H i s t o l o g i c a l studies, however, reveal that W/W  V  forming numerous microscopic  marrow i s capable of  colonies i n the spleens of i r r a d i a t e d  recipients (Lewis et a l . , 1967).  The colonies produced by W/W  as compared to those produced by +/+  marrow,  V  marrow, are fewer i n number, smaller  i n s i z e , slower to increase i n s i z e , and l a r g e l y myeloid rather than erythroid i n composition. a l l indicates that W/W  V  Nevertheless,  that they are produced at  marrow does contain a good supply of c e l l s  capable of hematopoietic d i f f e r e n t i a t i o n . growth rate of colonies derived from W/W  V  The reduced s i z e and slower c e l l s , however, "demonstrates  a d e f i n i t e r e s t r i c t i o n of p r o l i f e r a t i o n at early stages of c e l l ] d i f f e r e n t i a t i o n " (Lewis et a l . , 1967). may  [hematopoietic  The marrow of W/W  V  mice  contain nearly normal numbers of stem c e l l s , but the e f f e c t of  the W gene i s to make these c e l l s less able to multiply and t i a t e than normal c e l l s .  differen-  5  It  i s c l e a r t h a t n e i t h e r the s t e r i l i t y  pigment d e f e c t of W mutant mice i s secondary c r e s t d e f e c t has been demonstrated and non-mutant c e l l s v e r s i b l e by  to  characteristic  to the anemia:  the n e u r a l  i n v i t r o , where c o n d i t i o n s f o r mutant  are i d e n t i c a l ;  the s t e r i l i t y  the t w e l f t h day of embryonic l i f e ,  anomaly i s y e t apparent  nor the  i s maximal and  a l t h o u g h no  (Mintz and R u s s e l l , 1957).  irre-  hematopoietic  Each d e f e c t  appears  r e s u l t from some e f f e c t o f the W gene on a p a r t i c u l a r type o f  e i t h e r p r o s p e c t i v e pigment c e l l , stem c e l l .  The  p r i m o r d i a l germ c e l l ,  cell,  or hematopoietic  e x a c t n a t u r e o f the gene e f f e c t on each o f these  cell  t y p e s , however, i s unknown and i s v e r y much a s u b j e c t o f s p e c u l a t i o n . Both Mayer and Green (1968) and Bennett the a f f e c t e d c e l l  e_t a l . (1968) have n o t e d  types s h a r e the c h a r a c t e r i s t i c o f b e i n g m i g r a t o r y  and p r o l i f e r a t i v e ,  and have observed  t h a t i t i s m i g r a t i o n and  f e r a t i o n t h a t i s d e f i c i e n t i n each o f these c e l l mouse.  that  They have s u g g e s t e d  t h a t something  proli-  l i n e s i n the mutant  necessary  f o r these p r o -  cesses i s m i s s i n g o r a l t e r e d by the W gene. If  i n d e e d something  i s a l t e r e d by  f o r m i g r a t i o n and  proliferation  the W gene, then o t h e r m i g r a t o r y and p r o l i f e r a t i v e  might be a f f e c t e d , cyte, a c e l l  necessary  too.  Such a c e l l  cells  i n the a d u l t a n i m a l i s the lympho-  capable o f v e r y e x t e n s i v e m i g r a t i o n and,  s t i m u l a t i o n , marked t r a n s f o r m a t i o n and p r o l i f e r a t i o n .  under a p p r o p r i a t e To t e s t  the  h y p o t h e s i s t h a t the W gene i n t e r f e r e s w i t h m i g r a t i o n , a study was  made  v i n our l a b o r a t o r y to determine  i f the mutant W/W  l e s s c a p a b l e o f m i g r a t i o n than the normal +/+ Lymph node c e l l s  from e i t h e r W/W  V  lymphocyte was  lymphocyte  mice o r from t h e i r +/+  indeed  (Wong, 1969). littermates  6  were cultured on monolayers  of isogenic kidney c e l l s .  Migrating c e l l s  were followed with time-lapse cinemicrography and the rate of m o t i l i t y determined i n tracings from the micrographs.  +/+ lymphocytes on +/+  monolayers were found to move at an average rate of 10.8 u per minute, and W/W lymphocytes on W/W monolayers V  V  per minute.  at an average rate of 8.7 u  S t a t i s t i c a l analysis indicates that these rates are s i g n i -  ficantly different.  That W/W lymphocytes V  do move more slowly than  do +/+ lymphocytes suggests that something necessary f o r migration may indeed be the primary target of the W gene. In addition to an e f f e c t on migration, the W gene may also v v a f f e c t p r o l i f e r a t i o n i n lymphocyte l i n e s .  Spleens from W /W mice  have been found by plaque assay to produce one-third to one-half as many hemolysin-forming c e l l s as do spleens from W /+ mice (Shearer and Cudkowicz, 1967).  This defect could be explained i n three ways:  1) the presence of fewer antibody c e l l precursors i n W /W V  V  mice;  2) a decreased a b i l i t y of W /W antibody c e l l precursors to respond V  V  to the p r o l i f e r a t i v e stimulus; or 3) a decreased p r o l i f e r a t i v e p o t e n t i a l among i n d i v i d u a l antibody c e l l precursors.  Studies of hematopoiesis  have suggested that normal numbers of stem c e l l s are present i n W/W  V  mice (Lewis et a l . , 1967; Bennett e_t a l . , 1968).  Normal numbers of  antibody c e l l precursors are, therefore, also l i k e l y to be present. The defective hemolysin response of W/W mice i s most l i k e l y to be V  V  explained by some defect i n precursor c e l l p r o l i f e r a t i o n , s i m i l a r to the defect i n p r o l i f e r a t i o n exhibited by other hematopoietic stem c e l l s . The work of Wong (1969) and of Shearer and Cudkowicz (1967), then, i s not at variance with the hypothesis that something necessary  7  f o r m i g r a t i o n and p r o l i f e r a t i o n may be m i s s i n g o r a l t e r e d i n c e l l s o f W mice.  L e a v i n g , f o r the time b e i n g , s p e c u l a t i o n as t o the p r i m a r y  s i t e o f a c t i o n o f the W gene, I w i s h to c o n s i d e r the s i g n i f i c a n c e o f the e f f e c t o f such a gene on lymphocytes. to be the key c e l l s  Lymphocytes  a r e w e l l known  concerned w i t h i m m u n o l o g i c a l r e a c t i o n s o f a l l  including hypersensitivity  kinds,  r e a c t i o n s , a n t i b o d y s y n t h e s i s , and t i s s u e  r e j e c t i o n phenomena, and they appear t o be i n v o l v e d a t a l l s t a g e s o f these r e a c t i o n s , from the i n d u c t i o n o f the response to the f i n a l phase.  Indeed, lymphocytes a r e so i n t i m a t e l y i n v o l v e d i n a l l  of immune responses t h a t any i n t e r f e r e n c e m a n i f e s t e d by p r o f o u n d changes reactions.  phases  with their a c t i v i t i e s i s  i n the development  o f the v a r i o u s  immune  Many a s p e c t s o f these immune r e a c t i o n s , however, p a r t i c u l a r l y  of such complex phenomena as t i s s u e r e j e c t i o n , a r e i n c o m p l e t e l y stood.  effector  The e x i s t e n c e o f an a p p a r e n t l y abnormal lymphocyte  i n W mice prompted  under-  population  the s p e c u l a t i o n t h a t t h i s a b e r r a t i o n might be r e f l e c t e d  i n some a l t e r a t i o n o f immune phenomena i n these mice, and t h a t t h e s e mice might then p r e s e n t a u s e f u l i n v i v o system f o r the i n v e s t i g a t i o n o f those phenomena. The lymphocytes o f W mice appear on the average t o m i g r a t e somewhat more s l o w l y than lymphocytes from normal mice.  The d e f e c t i n  m i g r a t i o n i s a s m a l l one, b u t lymphocytes p l a y such a c e n t r a l r o l e i n immune responses t h a t any d e f e c t i n t h e i r a c t i v i t y , however s m a l l , might be markedly a m p l i f i e d o v e r the course o f an immune r e s p o n s e :  a small  d e f e c t i n m i g r a t i o n might i n f a c t have a l a r g e e f f e c t on r e a c t i o n s i n v o l v i n g the m i g r a t i n g c e l l s .  Slow-moving lymphocytes might, f o r  8  example, take l o n g e r to r e a c h p e r i p h e r a l a r e a s where i n t e r a c t i o n w i t h a n t i g e n may lymphoid  o c c u r , and they might  take l o n g e r to r e t u r n to c e n t r e s of  t i s s u e where t r a n s f o r m a t i o n and p r o l i f e r a t i o n a r e thought  o c c u r , thus p r o l o n g i n g the e n t i r e response t o a n t i g e n . lymphocytes  to  Conversely,  t h a t move more s l o w l y than normal might have g r e a t e r oppor-  t u n i t y f o r exposure o f lymphocytes  to and i n t e r a c t i o n w i t h a n t i g e n :  a g r e a t e r number  might become a c t i v a t e d and the response to a n t i g e n might  thus be enhanced. L i k e w i s e , a d e f e c t i n the p r o l i f e r a t i o n o f lymphocytes  from  W mice might be expected t o have p r o f o u n d e f f e c t s on the c o u r s e o f immune responses i n these mice.  Drugs which  i n t e r f e r e with  lymphocyte  p r o l i f e r a t i o n a r e commonly used to suppress immune r e s p o n s e s , i n d e e d , the W Similarly,  gene does appear to suppress the response t o SRBC a n t i g e n s .  the gene might  affect  as t i s s u e r e j e c t i o n , perhaps biting  and,  c e l l - m e d i a t e d immune responses  d e l a y i n g the r e j e c t i o n c r i s i s by  the p r o d u c t i o n o f lymphocytes  inhi-  s p e c i f i c f o r the f o r e i g n  The e x i s t e n c e of an a b e r r a n t lymphocyte  such  tissue.  p o p u l a t i o n i n an o t h e r -  w i s e i m m u n o l o g i c a l l y normal a n i m a l i s p o t e n t i a l l y o f g r e a t v a l u e i n immunological s t u d i e s .  Much i s known about lymphocytes  v i t i e s , b u t e x a c t l y how  these c e l l s p a r t i c i p a t e and behave a t v a r i o u s  s t a g e s o f immune effect  r e a c t i o n s i s not f u l l y  the d e s t r u c t i o n o f s o l i d  entirely  clear.  understood.  and t h e i r  How  acti-  lymphocytes  t i s s u e g r a f t s , f o r example, i s not  In v i v o s t u d i e s of immune phenomena such as  tissue  r e j e c t i o n o f t e n i n v o l v e such gross m a n i p u l a t i o n s of the immune as n e o n a t a l thymectomy, t h o r a c i c duct d r a i n a g e , treatment w i t h  system  9  immunosuppressant drugs, or even i r r a d i a t i o n , m a n i p u l a t i o n s which have v e r y b r o a d e f f e c t s on  immune r e s p o n s e s , e s s e n t i a l l y e l i m i n a t i n g  l e a s t some c a t e g o r i e s  o f lymphocyte a c t i v i t y  entirely.  l a t i o n s p r o v i d e a l i m i t e d amount of i n f o r m a t i o n  at  These manipu-  about the  functions  of lymphocytes, d e m o n s t r a t i n g , f o r example, t h a t c e r t a i n c l a s s e s lymphocytes and r e a c t i o n s , but cytes  during  c e r t a i n p r o c e s s e s are  e s s e n t i a l f o r p a r t i c u l a r immune  i n d i c a t i n g l i t t l e about the a c t i v i t i e s  the  course o f  of  those r e a c t i o n s .  of those lympho-  Attempts have been made  to study immune phenomena i n v i t r o — t h e mixed lymphocyte r e a c t i o n , example, i s c o n s i d e r e d  an i n v i t r o model f o r the p r o c e s s of  rejection—but  i n v i t r o models, i n e l i m i n a t i n g many of the  imposed by  i n v i v o s i t u a t i o n , are n e c e s s a r i l y  the  representation  of the  i n v i v o p r o c e s s , and  what a c t u a l l y happens i n v i v o . character  as  t i s s u e r e j e c t i o n c o u l d be  limited in their  not  accurately  represent  a l t e r e d i n some  the way the  to such complex phenomena  more r e a d i l y examined i n v i v o .  immunological p r o c e s s e s i n animals p o s s e s s i n g  could  variables  i m m u n o l o g i c a l background of the a n i m a l , then  c o n t r i b u t i o n o f lymphocyte a c t i v i t y  population  allograft  I f , however, a s i n g l e a s p e c t of  or b e h a v i o u r of lymphocytes c o u l d be  w i t h o u t a l t e r i n g the precise  may  for  an  aberrant  w i t h those p r o c e s s e s i n animals p o s s e s s i n g  Comparing  lymphocyte  normal lymphocytes  r e v e a l d i f f e r e n c e s which might i n d i c a t e j u s t what the s i g n i f i c a n t  events i n those p r o c e s s e s a r e . l e a d , in. ways we  can n o t  Studies  of abnormal r e a c t i o n s  often  p r e d i c t , to a b e t t e r u n d e r s t a n d i n g of normal  reactions. The  W mutant, then, appears e m i n e n t l y worthy o f i n v e s t i g a t i o n .  Experiments suggest a d e f e c t  i n migration  and  p r o l i f e r a t i o n of  10  lymphocytes  i n these mice, and i f these d e f e c t s c o u l d be shown to have  significant  e f f e c t s on immune phenomena, W mice might  prove  u s e f u l i n t h e f u r t h e r i n v e s t i g a t i o n o f those phenomena.  particularly  I propose,  t h e r e f o r e , t o examine some a s p e c t s o f b o t h c e l l u l a r and humoral immune responses i n W mice to determine what e f f e c t , i f any, the W gene e x e r t s on these r e a c t i o n s .  I n a d d i t i o n , I s h a l l attempt  t o c o n s i d e r the immuno-  l o g i c a l e f f e c t s o f t h i s l o c u s i n terms o f a h y p o t h e s i s f o r the p r i m a r y s i t e o f a c t i o n o f the W gene.  MATERIALS  I.  AND METHODS  MICE  WBB6-F1 h y b r i d b r e e d e r s o f W/+ and W /+ V  genotypes were o b t a i n e d  from the J a c k s o n L a b o r a t o r y i n August, 1970, and s u b s e q u e n t l y b r e d a t the  U n i v e r s i t y o f B r i t i s h Columbia t o produce h y b r i d s o f +/+, W/+,  W /+, V  and W/W  V  W/+ x W /+ V  genotype.  These mice, and t h e i r o f f s p r i n g produced by  b r o t h e r - s i s t e r b r e e d i n g , were used i n a l l experiments de-  scribed i n this  thesis.  Initial  s t u d i e s , however, which r e q u i r e d  +/+ mice, u t i l i z e d mice o f the C57B1/6J s t r a i n . to  Mice were always  only three  f i v e months o l d b e f o r e b e i n g s e l e c t e d f o r experiment. Mice o f the DBA/2J s t r a i n s e r v e d as t i s s u e donors i n a l l ex-  periments i n v o l v i n g a l l o g r a f t s . mice a t many l o c i ,  Mice o f t h i s s t r a i n d i f f e r from WBB6  i n c l u d i n g H-2, and DBA s k i n g r a f t s e l i c i t a s t r o n g  r e j e c t i o n response i n WBB6 h o s t s .  II.  SKIN GRAFT STUDIES  The r e j e c t i o n o f t r a n s p l a n t e d t i s s u e i n v o l v e s a complex  cell-  mediated immune r e s p o n s e , a response i n which lymphocytes p l a y a key role. or  Any i n t e r f e r e n c e w i t h normal lymphocyte movement, p r o l i f e r a t i o n ,  f u n c t i o n , as by s e v e r a n c e o f l y m p h a t i c d r a i n a g e from the g r a f t  d e p l e t i o n o f lymphocyte numbers, o r i n h i b i t i o n o f lymphocyte 11  cell  site,  12  division  c y c l e s , i s m a n i f e s t e d by  p r o c e s s and,  h i s t o l o g i c a l l y , by  infiltrating  the g r a f t .  An  to be  S k i n has studies, and  m a n i f e s t e d by l o n g been the  H loci.  Any  reject  difference  foreign  i n the  mice and  V  A preliminary mice, r a t h e r  V  A.  and  r e j e c t i o n process.  Skin  to be  of two  transplanted  a l s o appears to be  the  differences,  and  h o s t are matched at a l l groups of animals  m a n i f e s t most r e a d i l y i n  I chose t h e r e f o r e ,  to the  to i n v e s t i g a t e  their littermate controls  the  to s k i n a l l o g r a f t s .  s u g g e s t e d t h a t g r a f t r e j e c t i o n by  than b e i n g s l o w e r than normal, might i n f a c t be This  r e s u l t was  q u i t e unexpected, and  somethe  experiments were d e s i g n e d to determine i f indeed g r a f t  j e c t i o n i n W/W is  ability  experiment had  what f a s t e r than normal. following  cells  transplantation  to h i s t o c o m p a t i b i l i t y  tissue i s likely  response o f W/W  of the  ease w i t h which i t can be  i n d e f i n i t e l y u n l e s s donor and  response to s k i n a l l o g r a f t s .  V  a prolongation  s u b s e q u e n t l y examined f o r s u r v i v a l .  survive  i n the numbers of  t i s s u e of c h o i c e i n  l a r g e l y because o f the  w i l l not  tissue rejection  been suggested f o r the W gene, i s , t h e r e f o r e ,  most s e n s i t i v e of a l l t i s s u e s  W/W  a reduction  of the  i n h i b i t i o n o f lymphocyte m i g r a t i o n  p r o l i f e r a t i o n , such as has also l i k e l y  a prolongation  mice i s f a s t e r than i n +/+  mice, and  i f this  re-  difference  detectable h i s t o l o g i c a l l y .  Transplantation The  Technique  transplantation  i s b a s i c a l l y that described  t e c h n i q u e used throughout these by  Billingham  and  studies  Medawar (1951).  Mice which were to s e r v e as s k i n g r a f t donors were k i l l e d c e r v i c a l d i s l o c a t i o n , the  l e f t s i d e of  the  t h o r a x shaved, and  the  by skin  13  of  this  r e g i o n e x c i s e d and p l a c e d i n R i n g e r ' s  U s i n g a #11 fat,  scalpel,  solution in a Petri  the u n d e r s i d e of the s k i n was  s c r a p e d f r e e of muscle,  and b l o o d v e s s e l s , l e a v i n g o n l y the dermis and s u p e r f i c i a l  dermis i n t a c t .  T h i s s k i n was  cut i n t o two  epi-  o r t h r e e p i e c e s and each of  these trimmed to a r o u g h l y o v a l shape, about 1 cm x 3/4 The  dish.  g r a f t s were p l a c e d on f i l t e r paper moistened  cm i n s i z e .  with Ringer's  to  await  transplantation. Mice which were to r e c e i v e s k i n g r a f t s were a n a e s t h e t i z e d w i t h .005-.0075 ml p e r gram body weight of Nembutal (sodium p e n t o b a r b i t o l , 60 mg/ml) d i l u t e d 1:4 The  left  with Ringer's  s i d e o f the t h o r a x was  To form the g r a f t bed, fat  was  carefully  cm i n s i z e .  and a d m i n i s t e r e d  intraperitoneally.  shaved and s p r a y e d w i t h A e r o p l a s t .  the e p i d e r m i s , dermis,  and some subcutaneous  trimmed away from a r e g i o n a p p r o x i m a t e l y  1 cm x  The b l o o d v e s s e l s which s u p p l i e d t h i s a r e a formed a  network a c r o s s the g r a f t bed,  and were l e f t  intact.  3/4 fine  Figure 1 i l l u s -  t r a t e s normal mouse s k i n and the r e l a t i o n s h i p between g r a f t and  host  tissue. The  g r a f t i t s e l f was  s m a l l e r than the g r a f t bed, g r a f t was of  put i n p l a c e .  vaseline-impregnated  The  always the same s i z e as o r o n l y  slightly  and b o t h were d r i e d w i t h gauze b e f o r e g r a f t was  the  held i n place with a covering  gauze and a p l a s t e r bandage wrapped around  the body o f the mouse.  B.  Measurement of A l l o g r a f t The  determining  Survival  r e a c t i o n to a s k i n g r a f t can be measured most s i m p l y the s u r v i v a l time of the g r a f t on the h o s t .  A  by  comparison  14  Figure 1  Normal mouse s k i n . The e p i d e r m i s i s t h i n and d e n s e l y s t a i n e d . H a i r f o l l i c l e s ( h f ) and p i l o s e b a c e o u s glands (pg) p e n e t r a t e the dermis x200.  R e l a t i o n s h i p between g r a f t and h o s t t i s s u e . Panniculus adiposus and P a n n i c u l u s carnosus are removed from the g r a f t but r e t a i n e d i n t a c t i n the g r a f t bed.  15  }  E p i de  r m i s  Dermis  Panniculus adiposus  Panniculus carnosus  G R A F T  HOST 53  Epidermis Dermis Panniculus  adiposus  Panniculus  carnosus  16  of g r a f t s u r v i v a l times between e x p e r i m e n t a l  animals  and t h e i r c o n t r o l s  i s most s e n s i t i v e a t the time which corresponds  t o the LD^^ o r median  survival  The MST can be d e t e r -  time  (MST) ( B i l l i n g h a m e t a l . , 1954a).  mined by a p p l y i n g s k i n g r a f t s g r a f t s every  t o a number o f a n i m a l s ,  c h e c k i n g the  day f o r s i g n s o f r e j e c t i o n , and r e c o r d i n g the end p o i n t .  S t a t i s t i c a l procedures  can be a p p l i e d to the s u r v i v a l curve  to y i e l d  an e s t i m a t e o f the MST. Determination  o f the MST, however, r e q u i r e s o n l y t h a t the end  p o i n t o r day o f t o t a l r e j e c t i o n o f the g r a f t be r e c o r d e d ; i t r e q u i r e s s c o r i n g the .graft s i m p l y i n which the g r a f t s  dead o r a l i v e .  can be graded  On t h e o t h e r hand, a p r o c e d u r e  and g i v e n a n u m e r i c a l s c o r e  indicating  the e x t e n t o f the r e j e c t i o n p r o c e s s i n each o f them p r o v i d e s more i n f o r m a t i o n than a s i m p l e dead o r a l i v e c l a s s i f i c a t i o n , and i s t h e r e f o r e a more s e n s i t i v e measure than median s u r v i v a l time. i n my experiments  I t h e r e f o r e chose  t o make h i s t o l o g i c a l s t u d i e s o f s k i n  allografts,  s t u d i e s which would p e r m i t n o t o n l y a d e t e r m i n a t i o n o f the MST, b u t which would a l s o a l l o w comparisons t o be made between each  experimental  a l l o g r a f t and i t s c o n t r o l .  C.  Establishment  1) I s o g r a f t  o f C r i t e r i a by Which t o Measure A l l o g r a f t  Survival  Studies  In o r d e r t o determine  which changes i n an a l l o g r a f t  are asso-  c i a t e d w i t h the r e j e c t i o n p r o c e s s , those changes a s s o c i a t e d w i t h the wounding o f the s k i n or w i t h the h e a l i n g p r o c e s s must f i r s t be d e f i n e d .  Isografts are prepared and transplanted grafts, but heal i n permanently.  i n the same manner as are a l l o -  They are by d e f i n i t i o n i d e n t i c a l  with the tissue of the host animal, and w i l l not e l i c i t any r e j e c t i o n reaction.  Any changes i s o g r a f t s undergo can therefore be a t t r i b u t e d  only to the trauma of the transplantation procedure or to the healing process i t s e l f . Ten DBA/2 mice, males and females 9-12 weeks old, were grafted with skin from littermates of l i k e sex. a f t e r transplantation and continuing  Starting on the fourth day  through the tenth day, two or  three mice were k i l l e d on alternate days and the grafts prepared f o r h i s t o l o g i c a l study. 2) A l l o g r a f t Studies a) Determination of h i s t o l o g i c a l c r i t e r i a f o r assessing graft s u r v i v a l A preliminary study of skin a l l o g r a f t s on C57B1/6  allo-  mice was  undertaken i n order to determine the type and time course of h i s t o l o g i c a l changes occurring i n skin grafts undergoing r e j e c t i o n , and to e s t a b l i s h a standard set of c r i t e r i a f o r measuring the progress of graft destruction.  Eighty C57B1/6 mice, males and females 9-12 weeks  old, were grafted with skin from DBA/2 donors. always of l i k e sex. and continuing  Donor and host were  S t a r t i n g on the t h i r d day a f t e r transplantation  u n t i l the f i f t e e n t h day, f i v e or s i x mice were k i l l e d  each day and the grafts prepared f o r h i s t o l o g i c a l study as described below.  18 b)  D e t e r m i n a t i o n o f MST o f DBA/2 g r a f t s After  on C57B1/6 h o s t s  determining h i s t o l o g i c a l c r i t e r i a  of a l l o g r a f t r e j e c t i o n ,  the s l i d e s o f g r a f t  t i s s u e were  w i t h code numbers, r e o r d e r e d randomly, and s t o r e d They were then re-examined u s i n g the e s t a b l i s h e d on  a scale  for assessing  o f 1 t o 6 f o r degree o f r e j e c t i o n .  stages  relabelled  for several  months.  c r i t e r i a , and s c o r e d  For determination of  the MST, however, o n l y a dead o r a l i v e c l a s s i f i c a t i o n i s r e q u i r e d . allografts  receiving  g r a f t r e j e c t i o n s c o r e s o f 1, 2, 3, o r 4 were lumped  t o g e t h e r and c l a s s i f i e d " a l i v e , " and those r e c e i v i n g were c o n s i d e r e d "dead." on  D.  scores of 5 or 6  From t h e s e d a t a , t h e MST o f DBA/2 s k i n  C57B1/6 mice was determined, u s i n g a s i m p l i f i e d method o f  dose-response e f f e c t s  Thus  d e s c r i b e d by L i t c h f i e l d and W i l c o x o n  A l l o g r a f t R e j e c t i o n i n W/W  v  Mice: G r a f t s  Upon c o m p l e t i o n o f p r e l i m i n a r y  grafts  evaluating  (1948).  Exchanged A c r o s s H-2  studies  to e s t a b l i s h  Barriers  histolo-  g i c a l c r i t e r i a by which a l l o g r a f t r e j e c t i o n might be measured, two v experiments were d e s i g n e d t o compare s k i n with that  i n t h e i r +/+,. W/+,  and W /+ V  mice  littermates.  Preliminary h i s t o l o g i c a l studies grafts  g r a f t s u r v i v a l i n W/W  had i n d i c a t e d  that  DBA/2 s k i n  on +/+ mice underwent t h e i r most marked d e g e n e r a t i v e changes  between the s e v e n t h and e i g h t h days. p r o c e s s between +/+ and W/W  Any d i f f e r e n c e  mice, t h e r e f o r e was l i k e l y  apparent h i s t o l o g i c a l l y a t the e i g h t h day. i n which 34 W/W  V  t o be most  An experiment was d e s i g n e d  mice and 56 o f t h e i r +/+, W/+,  were g r a f t e d w i t h DBA/2 s k i n .  i n the r e j e c t i o n  and W /+ V  These mice were a l l k i l l e d  littermates on the  19  e i g h t h day a f t e r  transplantation,  and the s k i n  l o g i c a l study as d e s c r i b e d below.  histo-  on a s c a l e  code  o f 1 t o 6, u s i n g  c r i t e r i a d e s c r i b e d i n a) above. The  was  fixed for  The s l i d e s were l a b e l l e d w i t h  numbers and s c o r e d f o r degree o f r e j e c t i o n the  grafts  d i s t r i b u t i o n o f the g r a f t  compared w i t h t h a t  rejection  v o f W/+ and W /+ mice.  s c o r e s o f +/+ mice  A l s o , the d i s t r i b u t i o n v#  of the g r a f t  rejection  compared w i t h t h a t  s c o r e s o f +/+, W/+,  o f W/W  V  mice.  and W /+ mice t o g e t h e r was  The Mann-Whitney U - t e s t  ( S i e g e l , 1956)  was  used t o determine whether o r n o t t h e samples had been drawn from  the  same p o p u l a t i o n . A second experiment was d e s i g n e d t o measure the MST o f DBA/2  s k i n on t h e e x p e r i m e n t a l mice. one  o r two l i t t e r m a t e s  o f DBA/2 s k i n . tinuing  F o r t y - f i v e W/W  o f +/+, W/+,  o r W /+ V  day, a number o f W/W  V  t r o l s were k i l l e d each day and t h e s k i n  i n the s e c t i o n  genotype, r e c e i v e d  B e g i n n i n g on t h e s e v e n t h day a f t e r g r a f t i n g  through the t w e l f t h  examination.  mice, each p a i r e d  V  grafts  with  grafts  and con-  mice and t h e i r  con-  removed f o r h i s t o l o g i c a l  A l l o g r a f t s were p r e p a r e d f o r m i c r o s c o p y as d e s c r i b e d on h i s t o l o g i c a l t e c h n i q u e .  S l i d e s were l a b e l l e d w i t h  code numbers and s c o r e d f o r degree o f r e j e c t i o n c r i t e r i a established  earlier.  Allografts  were c o n s i d e r e d dead. M o r t a l i t y d a t a f o r DBA/2 s k i n  using the h i s t o l o g i c a l  receiving  scores of 5 or 6  a l l o g r a f t s on +/+, W/+,  W /+, V  v and  W/W  (1949).  mice were a n a l y z e d u s i n g t h e method o f L i t c h f i e l d Dose-effect straight  logarithmic-probability  and W i l c o x o n  l i n e s f o r each sample were p l o t t e d on  paper and the L D  s f )  o r MST read from the graph.  20 The  method a l l o w s d e t e r m i n a t i o n of 95%  MST  and  p e r m i t s an  samples.  The  estimation  of the h e t e r o g e n e i t y of  method a l s o p e r m i t s a comparison of  for  parallelism.  E.  A l l o g r a f t R e j e c t i o n i n W/W Mice: Histocompatibility Barriers V  Any  c o n f i d e n c e i n t e r v a l s f o r each  d i f f e r e n c e between +/+  Grafts  and W/W  the  a s l o w e r and  grafts.  Skin  short h e a l i n g - i n period, to b e l i e v e  that  is  r e l a t e d to the  within cells I t may  the can  a c r o s s H-2  then i s q u i c k l y  The  length  of the  lymph node, and  i n f i l t r a t e the  not be p o s s i b l e  g r a f t and  and  by  I f , on  the  f o r the  There i s no  reason  however,  speed of the  their destructive  s u r v i v a l time of  reaction  be  effects.  first-set  days, determined by  then, c o u l d not  two  the  grafts  healing-  rejection reaction  d e t e c t e d i n terms of  r e a c t i o n i t s e l f , then d i f f e r e n c e s between  c o n t r o l groups might be  Female mice w i l l carry  exert  8 to 10  for a  itself. MST.  o t h e r hand, g r a f t s u r v i v a l were p r o l o n g e d beyond the minimum  time r e q u i r e d mental and  only  speed w i t h which mononuclear  some r a t e - l i m i t i n g s t e p i n the  A stronger graft reaction,  incompatible  s h o u l d d i f f e r between the  to the  re-  a l l o g r a f t s which  acute r e j e c t i o n p e r i o d ,  to s h o r t e n the  beyond a c e r t a i n minimum, say in period  rejected.  a n t i g e n i c i t y o f the g r a f t , to the  draining  lines  mice i n speed of g r a f t  b a r r i e r s survives  the h e a l i n g - i n p e r i o d  groups of mice.  dose-effect  l e s s v i g o u r o u s response than do H-2  transplanted  two  Exchanged A c r o s s Weak  j e c t i o n might be more apparent i n a t e s t u t i l i z i n g incite  the MSTs of  a Y-linked  more apparent.  reject skin grafts  histocompatibility  experi-  antigen  from c o i s o g e n i c (Silvers,  males which  1968).  Rejection  across this weak h i s t o c o m p a t i b i l i t y b a r r i e r takes several weeks, and may  therefore provide a more s e n s i t i v e system for measuring small d i f -  ferences between g r a f t r e j e c t i o n times i n d i f f e r e n t groups of mice. Skin grafts from male +/+  mice were prepared as described  (p. 12) and transplanted to female +/+ one +/+  mice and s i x W/W  V  or W/W  V  l i t t e r m a t e hosts.  mice received g r a f t s .  earlier Twenty-  Grafts were removed  17 to 24 days a f t e r transplantation, prepared f o r h i s t o l o g i c a l a n a l y s i s , and scored b l i n d l y for degree of r e j e c t i o n using the h i s t o l o g i c a l c r i t e r i a established e a r l i e r .  F.  H i s t o l o g i c a l Preparation of Skin Grafts Mice were k i l l e d by c e r v i c a l d i s l o c a t i o n and the g r a f t s , with  a narrow border of host skin, were excised, f i x e d i n Allen's B-15 hour at 37°C), and subsequently stored i n Bouin's f i x a t i v e 1963).  Allen's B-15  (1  (Culling,  i s a superior nuclear f i x a t i v e (Baker, 1958)  and  seemed to give generally more s a t i s f a c t o r y r e s u l t s than Bouin's alone. Tissue was  dehydrated, embedded i n Paraplast, sectioned at 7 or 8 p,  and stained with eosin and E h r l i c h ' s haematoxylin according to procedures described i n C u l l i n g (1963).  III.  LYMPHOCYTE ACTIVATION STUDIES  The a l l o g r a f t response involves the stimulation and p r o l i f e r a t i o n of a population of c i r c u l a t i n g lymphocytes.  The number of such activated  c e l l s i n the peripheral blood, as measured by the uptake of radioactive  22  DNA p r e c u r s o r s  such as t r i t i a t e d  thymidine, i s c o n s i d e r e d  i n d i c a t o r o f the imminence o f g r a f t r e j e c t i o n .  a reliable  The f o l l o w i n g  experi-  ments were d e s i g n e d t o determine whether o r n o t g r a f t r e j e c t i o n i n W/W  mice i s a s s o c i a t e d w i t h a g r e a t e r number o f a c t i v a t e d c e l l s i n  V  the p e r i p h e r a l b l o o d W/+,  A.  o r W /+  o f these mice than i s the g r a f t r e j e c t i o n o f +/+,  controls.  V  Labelling i n vivo Ten W/W  mice, males and females, each p a i r e d w i t h a +/+,  V  o r W /+ V  W/+,  l i t t e r m a t e o f l i k e sex, were g r a f t e d w i t h s k i n from DBA/2 donors.  Four hours b e f o r e  t h e mice were t o be k i l l e d on t h e s e v e n t h ,  nineth,  days a f t e r t r a n s p l a n t a t i o n , they were i n j e c t e d i n t r a -  and t e n t h  p e r i t o n e a l l y w i t h 1 yC p e r gram body weight o f t r i t i a t e d (S.A.  20 Ci/mM, ICN, C a l i f o r n i a ) .  eighth,  thymidine  Mice were b l e d from the o r i b i t a l  s i n u s w i t h 50X c a p i l l a r y p i p e t t e s , and .05 ml from each mouse was p r e pared f o r s c i n t i l l a t i o n mice and two W/W  V  as u n t r e a t e d  B.  counting  (p. 2 3 ) . F i v e +/+, W/+,  and W /+ V  mice which h a d n o t been g r a f t e d were a l s o sampled  controls.  Labelling i n vitro Ten W/W  V  mate o f l i k e sex, of W/W  V  mice, male and female, each p a i r e d w i t h a +/+ r e c e i v e d DBA/2 s k i n g r a f t s .  and +/+ mice r e c e i v e d a u t o g r a f t s .  fourteenth  litter-  Two a d d i t i o n a l p a i r s  On t h e f o u r t h , e i g h t h , and  days a f t e r t r a n s p l a n t a t i o n , b l o o d  samples were taken from  each o f t h r e e p a i r s o f a l l o g r a f t e d mice; the a u t o g r a f t e d  p a i r s were  sampled on not  days f o u r and  Samples of  W/W  .1 ml were p l a c e d  i n 12  o f t i s s u e c u l t u r e medium and  f i l m and period,  and  v  f o u r +/+  untreated  x 100 1 uC  mm  tritiated  f o r each mouse.  i n a c t i v a t e d h o r s e serum,  At  the end  Tritiated  thymidine  (S.A.  C.  Scintillation  15  v i v o or i n v i t r o ,  culture  i n 12  centrifuged  a t 800  s u p e r n a t a n t was  x 100 g  mm  glass  t e s t tubes.  been l a b e l l e d i n v i v o  In v i t r o  f o r 10 minutes to sediment the To each sample was  i n 10%  s a l i n e ) , and  Samples were p r e c i p i t a t e d w i t h 3 ml  in a clinical  and  thymidine e i t h e r i n  Samples o f b l o o d which had  removed.  The  streptomycin,  samples o f whole b l o o d were p r e p a r e d f o r l i q u i d s c i n -  c a r r i e r (2% BSA  hour.  15%  Counting  counting.  were p l a c e d  rpm  o f the  Ci/mM, s t e r i l e s o l u t i o n i n water)  A f t e r f o u r hours exposure to t r i t i a t e d  f o r 1/2  Triplicate  counting.  L-glutamine, p e n i c i l l i n ,  o b t a i n e d from ICN, C a l i f o r n i a .  ml  thymidine.  of E a g l e ' s medium supplemented w i t h  was  .03  containing  t i s s u e c u l t u r e medium used i n a l l experiments i n t h i s s e r i e s  Dulbecco's m o d i f i c a t i o n  tillation  t e s t tubes  pipettes.  Tubes were c l o s e d w i t h P a r a -  c e l l s were h a r v e s t e d f o r s c i n t i l l a t i o n  heparin.  had  controls.  glass  i n c u b a t e d f o r f o u r hours a t 37°C.  The  mice which  from the o r i b i t a l s i n u s w i t h 50 A c a p i l l a r y  c u l t u r e s were e s t a b l i s h e d  was  Two  been g r a f t e d were a l s o sampled as Mice were b l e d  1 ml  eight.  p r e c i p i t a t e was centrifuge  added .09  ml  c o l d 5% TCA sedimented by  c u l t u r e s were  cells,  .03  ml  and  cold  the thymidine,  sodium l a u r y l  and  i n c u b a t e d at  spinning  sulphate. 4°C  at 2,500  f o r ten minutes; the s u p e r n a t a n t was  then  24  removed. TCA  The p e l l e t was resuspended  and 3 ml c o l d a b s o l u t e e t h a n o l .  and washed twice w i t h 3 ml c o l d A f t e r the l a s t wash, 1 ml NCS  t i s s u e s o l u b i l i z e r was added t o the p e l l e t , and t h e tubes were s e a l e d and i n c u b a t e d a t 37°C o v e r n i g h t . to s c i n t i l l a t i o n scintillation  vials  fluid.  scintillation vials fluid,  The samples were then  and t h e tubes  rinsed  transferred  t h r e e times w i t h  1.5 ml  These washings were added to t h e samples i n the and the volume made up t o 15 ml w i t h  scintillation  a t o l u e n e s c i n t i l l a t o r s o l u t i o n c o n t a i n i n g 6 g/1 PPO and .75  mg/1 POPOP.  V i a l s were s t o r e d i n t h e dark a t 6°C f o r 24 hours  c o u n t i n g i n a N u c l e a r Chicago  Isocap  300 l q u i d s c i n t i l l a t i o n  before  counter.  Each sample was counted  f o r t h r e e 40 minute i n t e r v a l s , and t h e counts  averaged.  r a t i o method was used t o determine  The channels  counting  e f f i c i e n c y , and r e s u l t s a r e e x p r e s s e d as d i s i n t e g r a t i o n s p e r minute,  IV.  HUMORAL IMMUNE RESPONSES  The humoral immune response  i n v o l v e s the p r o d u c t i o n o f c i r c u -  l a t i n g antibodies to c e r t a i n foreign antigens.  Mice respond  t o an  i n j e c t i o n o f f o r e i g n r e d b l o o d c e l l s by p r o d u c i n g a n t i b o d i e s c a p a b l e of a g g l u n t i n a t i n g t h e i n j e c t e d c e l l s , o f l y s i n g them.  o r , i n the presence  The p r o d u c t i o n o f h e m a g g l u t i n i n s  by s t a n d a r d s e r i a l d i l u t i o n of c i r c u l a t i n g antibody.  o f complement,  can be monitored  t e c h n i q u e s which i n d i c a t e the serum  The p r o d u c t i o n o f h e m o l y s i n s  most s e n s i t i v e l y by a t e c h n i q u e which p e r m i t s a l l y p r o d u c i n g a n t i b o d i e s to be e s t i m a t e d .  titer  can be monitored  the number o f c e l l s a c t u -  The f o l l o w i n g  experiments  25  were d e s i g n e d to determine whether any d i f f e r e n c e e x i s t s between +/+ and W/W  mice i n t h e i r a b i l i t y  V  t o produce  a g g l u t i n a t i n g or l y t i c  b o d i e s i n response to s t i m u l a t i o n w i t h sheep  A.  P r o d u c t i o n o f SRBC A g g l u t i n i n s i n +/+ F i v e W/W  V  mice and t h e i r +/+  red blood  and W/W  v  anti-  cells.  Mice  l i t t e r m a t e s were immunized w i t h  an i n t r a p e r i t o n e a l i n j e c t i o n o f .1 ml o f 20% SRBC i n Dulbecco's b a l a n c e d salt solution.  To i n d u c e a secondary r e s p o n s e , mice were i n j e c t e d a g a i n  18 days a f t e r the immunizing  dose.  Mice were b l e d from the o r b i t a l s i n u s b e f o r e immunization a g a i n on the 4 t h day a f t e r immunization, and a t r e g u l a r i n t e r v a l s this.  Mice were s t a r v e d f o r 12 t o 16 hours b e f o r e b l e e d i n g .  microliters hematocrit  after  Fifty  o f b l o o d were c o l l e c t e d a t each sampling time i n m i c r o capillary  a t room temperature  tubes.  B l o o d was a l l o w e d t o c l o t  and then f o r f o u r hours a t 4°C.  c e n t r i f u g e d a t 15,000 g f o r f i v e minutes centrifuge,  (day 0 ) ,  f o r one hour  The tubes were  i n an Adams M i c r o - h e m a t o c r i t  then broken j u s t above the i n t e r f a c e between the serum and  the c l o t , and the serum t r a n s f e r r e d t o a c l e a n c a p i l l a r y were then i n c u b a t e d f o r t h i r t y minutes a c t i v a t e complement, and f i n a l l y  tube.  Sera  i n a water b a t h a t 60°C t o i n -  s t o r e d a t 4°C f o r 24 hours b e f o r e  testing. Mouse s e r a were t e s t e d f o r the p r e s e n c e o f SRBC a g g l u t i n i n s by the d i r e c t h e m a g g l u t i n a t i o n method.  A micro-method u s i n g d i s p o s a b l e  p l a s t i c m i c r o t i t e r p l a t e s and m i c r o l i t e r volumes o f the r e a c t a n t s was employed.  Appropriate dilutions  (lOx, o r lOOx and 150x) o f each serum  26  sample i n Dulbecco's BSS were prepared.  50  u  l of BSS were placed i n  each w e l l except the f i r s t i n a series of eight wells.  50 M l of serum  of the d i l u t i o n to be tested were placed i n the f i r s t w e l l and i n the second w e l l .  The contents of the second w e l l were mixed by p i p e t t i n g ,  and 50 y l transferred to the t h i r d well.  In this manner, s e r i a l two-  f o l d d i l u t i o n s were made through seven wells. w e l l were discarded,  50 y l from the seventh  leaving the eighth w e l l as a control with no serum.  Two series of two-fold  d i l u t i o n s were prepared for each serum sample  tested, one series s t a r t i n g with a serum d i l u t i o n of lOOx and the other s t a r t i n g v/ith a serum d i l u t i o n of 150x. then added to each w e l l .  50 y l of .4% SRBC i n BSS were  The plates were incubated f o r 3 hours at  room temperature and then overnight  at 4°C. The degree of hemagglu-  t i n a t i o n i n each w e l l was scored as ++, +, +-, or - according  to the  following c r i t e r i a : +-  granular f i l m of c e l l s surrounding small button or ring of c e l l s  + or ++  The  smooth-edged button of c e l l s i n center of w e l l  d i f f u s e f i l m of agglutinated well  c e l l s covering bottom of  t i t e r of the serum was taken to be the highest  d i l u t i o n giving  d e f i n i t e agglutination, that i s , scoring ++ or +.  B.  Production of Plaque-forming C e l l s i n +/+, W/+, W/+, V  Mice of +/+, W/+, W/+, V  and W/W  V  and W/W  V  Mice  genotypes (four mice of each  type) were immunized with an i n t r a p e r i t o n e a l i n j e c t i o n of .1 ml of 20% SRBC i n Dulbecco's balanced s a l t s o l u t i o n .  Four or s i x days a f t e r  27  immunization  a l l mice were k i l l e d by c e r v i c a l d i s l o c a t i o n and the s p l e e n s  removed t o Syracuse d i s h e s c o n t a i n i n g 5 ml BSS. rubbed  a g a i n s t a w i r e mesh and thereby broken  of c e l l s ;  Each s p l e e n was g e n t l y  a p a r t to r e l e a s e  clumps  these clumps were then f u r t h e r d i s p e r s e d by a s p i r a t i n g t h e  s u s p e n s i o n r e p e a t e d l y through a t u b e r c u l i n s y r i n g e f i t t e d w i t h a 25 gauge n e e d l e .  The s u s p e n s i o n was then removed t o a c e n t r i f u g e  and i n c u b a t e d i n an i c e b a t h f o r 10-15 minutes clumps o f c e l l s  t o a l l o w any r e m a i n i n g  t o s e t t l e o u t , a f t e r which the lower 1-2 ml o f the  s u s p e n s i o n was removed w i t h a P a s t e u r p i p e t t e .  The r e m a i n i n g  s u s p e n s i o n was c e n t r i f u g e d a t 1,200 g f o r 10 minutes, removed, and the c e l l s  resuspended  c e l l counts were performed the s u s p e n s i o n d i l u t e d  the supernatant  At t h i s s t a g e ,  total  t o an a p p r o p r i a t e c o n c e n t r a t i o n (about 2 x 1 0  6  tests.  .35 ml o f each s p l e e n c e l l s u s p e n s i o n was added .1 ml o f  a 10% s o l u t i o n o f washed, packed p i g serum.  i n 5 ml BSS.  cell  i n a hemocytometer, and a s m a l l volume o f  c e l l s / m l ) f o r use i n the f o l l o w i n g To  tube  SRBC and .05 ml o f u n d i l u t e d  A f t e r thorough m i x i n g ,  i n each o f t h r e e PFC p l a t e s  guinea  .1 ml o f t h i s m i x t u r e was p l a c e d  (see b e l o w ) .  The p l a t e s were s e a l e d w i t h  m e l t e d v a s e l i n e and i n c u b a t e d a t 37°C f o r 1 1/2 h o u r s . were p r e p a r e d f o r each mouse t e s t e d .  Three  plates  C l e a r areas o r plaques were p l a i n l y  v i s i b l e and e a s i l y counted when the p l a t e s were observed a g a i n s t a light. The PFC p l a t e s used i n t h e above t e s t s have been d e s c r i b e d by Cunningham (1968a). in  O r d i n a r y g l a s s microscope  70% a l c o h o l and d r i e d .  s l i d e s were washed  A t each end o f one s l i d e was p l a c e d a p i e c e  28  of S c o t c h b r a n d tape which c a r r i e d a d h e s i v e on e i t h e r s i d e ; each p i e c e o f tape was  1/2  i n c h wide and l o n g enough t o span  A second s l i d e was  then p l a c e d on top o f the f i r s t ,  r e g i s t e r so t h a t a narrow l i p was (Figure 2b).  The  pieces of s t i c k y o f about  .1 ml.  the s l i d e  left  slightly  out of  a l o n g the l o n g edge o f the  chamber thus c r e a t e d between the two tape was  (Figure 2a).  a p p r o x i m a t e l y one  slides  A f t e r adding a s u s p e n s i o n of s p l e e n c e l l s ,  SRBC, and  by  MITOGEN STIMULATION STUDIES  response o f lymphocytes  the response o f t h e s e c e l l s  to a n t i g e n s i s mimicked i n v i t r o  to l i p o p o l y s a c c h a r i d e  (LPS) from E_. c o l i  and t o v a r i o u s p l a n t l e c t i n s such as p h y t o h e m a g g l u t i n i n mitogen  (PWM) , and C o n c a n a v a l i n - A  lymphocyte  (Con-A).  (PHA), pokeweed  These substances  induce  t r a n s f o r m a t i o n and p r o l i f e r a t i o n s i m i l a r to t h a t seen i n  o t h e r c i r c u m s t a n c e s where lymphocytes to f o r e i g n a n t i g e n s .  to s t i m u l a t e DNA  a r e b e l i e v e d to be  D i f f e r e n t mitogens  p o p u l a t i o n s o f lymphocytes.  DNA  each  vaseline.  V.  The  the  c e l l deep, and had a volume  g u i n e a p i g complement, the chamber c o u l d be s e a l e d by c o a t i n g l i p w i t h melted  and  slide  In mice,  synthesis i n B cells  appear  to a c t i v a t e  different  f o r example, LPS has been shown o n l y , and Con-A t o s t i m u l a t e  s y n t h e s i s i n T c e l l s o n l y (Andersson e t a l . , 19 72a).  then, p r o v i d e a means o f s e l e c t i v e l y  responding  i n d u c i n g DNA  These  mitogens,  synthesis i n T - c e l l  o r B - c e l l p o p u l a t i o n s ; the magnitude o f the response  t o the two  mitogens  p e r m i t s an e s t i m a t i o n of the r e l a t i v e numbers o f r e s p o n d i n g T- and  B-  29  S c o t c h d o u b l e - s t i c k tape i s p l a c e d a t e i t h e r end of a p l a i n microscope s l i d e .  A second microscope s l i d e i s p l a c e d on top of the f i r s t , s l i g h t l y out o f r e g i s t e r . The space between the two s l i d e s i s about .1 ml i n volume and can be f i l l e d by c a p i l l a r y ' action. The chamber i s s e a l e d by p a i n t i n g the l i p s w i t h m e l t e d vaseline.  C o n s t r u c t i o n o f PFC p l a t e s .  30  cells  i n a mixed p o p u l a t i o n .  The f o l l o w i n g experiment was d e s i g n e d  to t e s t the responses o f +/+ and W/W  s p l e e n c e l l s , which  V  p o p u l a t i o n o f T- and B - c e l l s ,  to the mitogens  a r e a mixed  Con-A and LPS, and thereby  to determine i f the r e l a t i v e numbers o f r e s p o n s i v e T- and B - c e l l s a r e the same i n the two groups o f mice. The f o l l o w i n g p r o c e d u r e s were c a r r i e d out under s t e r i l e  con-  ditions. Mice were k i l l e d by c e r v i c a l d i s l o c a t i o n and t h e s p l e e n s removed to S y r a c u s e d i s h e s c o n t a i n i n g 5 ml o f Dulbecco's b a l a n c e d s a l t U s i n g n e e d l e p r o b e s , the organs were t e a s e d a p a r t to r e l e a s e of c e l l s .  solution.  clumps  These were d i s p e r s e d by a s p i r a t i n g the s u s p e n s i o n r e p e a t e d l y  i n and out o f a t u b e r c u l i n s y r i n g e f i t t e d w i t h a 25 gauge n e e d l e .  The  s u s p e n s i o n o f s i n g l e c e l l s was then c e n t r i f u g e d f o r 10 minutes a t 1,500 g, the s u p e r n a t a n t removed, and the c e l l s containing a n t i b i o t i c s  resuspended i n 5 ml o f RPMI  and 10% f e t a l c a l f serum.  Total c e l l  counts  were performed i n a hemocytometer and the c e l l s u s p e n s i o n i n each a d j u s t e d to a c o n c e n t r a t i o n o f 2.5 x 1 0 Mitogen s t i m u l a t i o n  c e l l s p e r ml..  t e s t s were performed i n f l a t - b o t t o m e d d i s -  posable p l a s t i c m i c r o t i t e r p l a t e s  ( L i n b r o ISFB 96-TC) a t e c h n i q u e adapted  from Hartzman e t a l . (19 71) by P e a r s o n were d e l i v e r e d by a u t o m a t i c p i p e t t e well.  6  tube  (19 74).  20 ml o f c e l l s u s p e n s i o n  (Hamilton Co. //PB-600-1) i n t o  each  .05 ml o f mitogen, e i t h e r C o n c a n a v a l i n - A o r L i p o p o l y s a c c h a r i d e ,  a t v a r i o u s c o n c e n t r a t i o n s , were added of RPMI was added  t o the c e l l s u s p e n s i o n s ; .05 ml  to the c o n t r o l c u l t u r e s .  f o r each c u l t u r e c o n d i t i o n .  Four r e p l i c a t e s were p r e p a r e d  The p l a t e s were i n c u b a t e d a t 48, 72, o r  31  96 hours  a t 37°C i n a C 0 - e n r i c h e d atmosphere. 2  harvesting,  .05 ml (1 u C i ) o f t r i t i a t e d  England N u c l e a r ) was added t o each  16 t o 18 hours  thymidine  before  (S.A. 2Ci/mM, New  well.  C e l l s were h a r v e s t e d by a s p i r a t i n g the c o n t e n t s o f each  well  through a m i l t i - s a m p l e m i c r o - h a r v e s t e r which drew the c e l l s onto o f g l a s s f i b e r f i l t e r paper  (Pearson, 1974).  Each f i l t e r  discs  d i s c was  washed w i t h 10 ml o f d i s t i l l e d water and 5 ml o f acetone, p l a c e d i n a scintillation vial, tillation  and d r i e d a t 110°C f o r 10 minutes.  f l u i d were added t o each v i a l .  N u c l e a r Chicago Isocap  300 s c i n t i l l a t i o n  The v i a l s were p l a c e d i n a c o u n t e r and a l l o w e d to e q u i l i -  b r a t e f o r 12 t o 24 hours b e f o r e c o u n t i n g . 10  Each v i a l was counted f o r  minutes.  VI.  A.  5 ml o f s c i n -  OTHER STUDIES  Macrophage M i g r a t i o n S t u d i e s Macrophages a r e a c t i v e m i g r a t o r y c e l l s  and might be e x p e c t e d  to be a f f e c t e d by a gene which i n h i b i t s m i g r a t i o n i n some c e l l The  f o l l o w i n g experiment  lines.  was d e s i g n e d t o determine whether o r n o t t h e  W gene has any g r o s s e f f e c t on the m i g r a t o r y r a t e s o f mouse macrophages. Mice were i n j e c t e d i n t r a p e r i t o n e a l l y w i t h 3 ml s t e r i l e m i n e r a l 011 by  to induce p e r i t o n i t i s .  Three  days l a t e r ,  these mice were  killed  c e r v i c a l d i s l o c a t i o n and i n j e c t e d i n t r a p e r i t o n e a l l y w i t h 5 ml  Dulbecco's  balanced s a l t  solution.  As much f l u i d as p o s s i b l e was w i t h -  drawn from the body c a v i t y , and an i n c i s i o n  then made i n the body w a l l .  32  The body c a v i t y was to  the f i r s t .  at  800  washed w i t h another 3 ml BSS,  The s u s p e n s i o n o f c e l l s  g f o r 10 minutes;  medium and capillary  finally tubes  thus o b t a i n e d was  end.  The  packed  centrifuged  the c e l l s were washed once w i t h  resuspended  Dulbecco's  i n 2 o r 3 drops o f the medium.  (Drummond) were f i l l e d w i t h the c e l l  s e a l e d a t one end by h e a t i n g i n a Bunsen flame. t r i f u g e d a t 800  and t h i s wash added  g f o r 10 minutes  s u s p e n s i o n and  These  to pack the c e l l s  20 X  tubes were cen-  i n t o the s e a l e d  tubes were then broken j u s t below the i n t e r f a c e between the  cells  and the s u p e r n a t a n t t o y i e l d a c u l t u r e tube 3-5  and f i l l e d w i t h packed i n Mackaness-type  cells.  chambers  These  mm  long  c u l t u r e tubes were then p l a c e d  (Figure 3).  Two  c o n t r o l and two e x p e r i m e n t a l  c u l t u r e tubes were p l a c e d i n each chamber and h e l d i n p l a c e w i t h dabs of  s i l i c o n e grease.  The  chamber was  then f i l l e d w i t h Dulbecco's medium,  s e a l e d w i t h p a r a f f i n , and i n c u b a t e d a t 37°C f o r 8 h o u r s . of  At the end  the i n c u b a t i o n p e r i o d , the chambers were p l a c e d on the s t a g e o f a  microscope f i t t e d w i t h a p r o j e c t i o n e y e p i e c e .  The shadows o f the  cul-  t u r e tubes w i t h i n the chamber and o f the a r e a c o v e r e d by the m i g r a t i n g c e l l s were c l e a r l y p r o j e c t e d on a s m a l l s c r e e n p l a c e d n e a r the m i c r o scope and c o u l d be r e c o r d e d by The a r e a c o v e r e d by meter.  V  B.  the m i g r a t i n g c e l l s was  P e r i t o n e a l exudate  and ten W/W  t r a c i n g the o u t l i n e s o f the shadows.  cells  then measured w i t h a p l a n i -  from seven +/+,  t h r e e W/+,  five  mice were t e s t e d .  Lymphocyte Adhesion S t u d i e s Mice were k i l l e d by  c e r v i c a l d i s l o c a t i o n and the p o p l i t e a l  lymph nodes removed to a Syracuse d i s h c o n t a i n i n g 3 ml balanced s a l t  solution.  Dulbecco's  U s i n g n e e d l e p r o b e s , the nodes were t e a s e d  33  T h i s chamber c o n t a i n s two c u l t u r e tubes h e l d i n p l a c e w i t h s i l i c o n e grease, and shows the a r e a covered by c e l l s m i g r a t i n g from each tube. The chamber i s s e a l e d w i t h round cover s l i p s and f i l l e d w i t h medium v i a a s y r i n g e i n s e r t e d through a s m a l l h o l e i n one s i d e . This hole i s s e a l e d w i t h m e l t e d wax.  F i g u r e 3.  Mackaness-type chamber f o r c e l l - m i g r a t i o n studies.  34  apart to release clumps of c e l l s ; the clumps were further broken up by a s p i r a t i n g the suspension repeatedly  i n and out of a tuberculin  syringe f i t t e d with a 26 gauge needle.  The suspension of s i n g l e c e l l s  thus obtained was cells.  centrifuged at 800 g for 10 minutes to sediment the  The supernatant was  removed and the c e l l p e l l e t resuspended  in 2 ml Dulbecco's medium supplemented with 15% horse serum, a n t i b i o t i c s , and L-glutamine.  At this stage, t o t a l c e l l counts were performed i n  a hemocytometer and the c e l l concentration 6 x 10  5  c e l l s per  adjusted to approximately  ml.  Culture chambers were constructed by attaching glass rings (15 mm  i n t e r n a l diameter, 5 mm  i n height) to ordinary glass microscope  s l i d e s with s i l i c o n e grease (Figure 4a). each s l i d e .  Two  rings were attached  About 1 ml of c e l l suspension was  to  placed i n each chamber  and the chamber sealed with s i l i c o n e grease and glass cover s l i p s which had been precleaned  i n 70% alcohol (Figure 4b).  then inverted (Figure 4c), and were incubated  The chambers were  for 30 minutes at 37°C.  At the end of the incubation period, the number of lymphocytes per unit area which had s e t t l e d on each cover s l i p was an inverted microscope.  determined using  Using a 40x objective and a g r i d placed i n a  lOx ocular, the number of c e l l s i n twenty d i f f e r e n t f i e l d s , ten f i e l d s on a v e r t i c a l axis and ten on a horizontal axis, were counted. average count per f i e l d was was  The  calculated for each culture chamber, and  usually about 30 to 50 c e l l s .  After determining the average number  of c e l l s per f i e l d that were resting on the cover s l i p s , the chambers were righted (Figure 4d).  C e l l s which had not adhered to the glass  35  G l a s s r i n g s a r e a t t a c h e d to o r d i n a r y microscope s l i d e s w i t h s i l i c o n e grease s p r e a d round one r i m o f the ring.  *P|  Each r i n g i s f i l l e d w i t h c e l l s u s p e n s i o n and s e a l e d w i t h a cover s l i p and s i l i c o n e g r e a s e . c.  The chamber i s i n v e r t e d . The c e l l s s e t t l e on the cover s l i p and can be counted w i t h an i n v e r t e d microscope. The chamber i s r i g h t e d . The c e l l s a d h e r i n g to the cover g l a s s can be counted u s i n g a r e g u l a r m i c r o scope.  F i g u r e 4.  R i n g chambers f o r c e l l a d h e s i o n s t u d i e s  i  • fif^T  36 cover s l i p s f e l l away, but those which had become attached could be counted using a regular microscope.  Again, using a 40x objective and  a g r i d placed i n a lOx ocular, the number of c e l l s i n twenty d i f f e r e n t f i e l d s were counted, and the average count per f i e l d calculated f o r each culture chamber.  The number of adherent c e l l s i s expressed as  a percentage of the number of c e l l s o r i g i n a l l y resting on the glass cover s l i p .  Lymph node c e l l s from seven +/+  and seven W/W  V  mice were  tested.  VII.  HAEMATOLOGICAL TECHNIQUES  Total and d i f f e r e n t i a l white blood c e l l counts were performed on ungrafted adult mice and on mice bearing skin a l l o g r a f t s or skin v* autografts.  At l e a s t eight mice of each genotype, +/+,  W/+,  W /+, and  v W/W  were sampled i n each category.  Mice were bled from the o r b i t a l  sinus using heparinized c a p i l l a r y tubes.  Because the sampling process  i t s e l f can a f f e c t l a t e r counts, each mouse was bled once only. haematological techniques were used throughout:  Standard  t o t a l counts were'  performed using an improved Neubauer chamber; blood films were stained with Wright's blood s t a i n .  RESULTS  I.  A.  H i s t o l o g i c a l Features  SKIN GRAFT STUDIES  of S k i n I s o g r a f t  Healing  Except f o r minor, t r a n s i t o r y a b n o r m a l i t i e s ,  isografts retain  the appearance of normal s k i n throughout the h e a l i n g - i n p e r i o d . dermal h y p e r p l a s i a , dermal oedema, and  round c e l l i n f i l t r a t i o n ,  never severe,  a r e maximal a t about the s i x t h day  By  day,  the e i g h t h  normal s k i n .  The  marized i n Table The  I and  i l l u s t r a t e d i n Figure  5.  appearance of numerous f i b r o b l a s t s i n the g r a f t bed the  f o u r t h day  of g r a n u l a t i o n t i s s u e .  c a p i l l a r i e s i n these a r e a s and graft i s healing i n . morphonuclear c e l l s , stage o f t e n separates  the s i x t h  accompanies t h i s p r o c e s s .  The  new  that  epidermis at  from the dermis d u r i n g h i s t o l o g i c a l  day,  cellular  This i n f i l t r a t e  morphonuclear c e l l s ,  A p r o l i f e r a t i o n of  Very m i l d c e l l u l a r i n f i l t r a t i o n , m a i n l y of  and  polythis  preparation,  of the g r a f t appears  greater  than at day  c o n s i s t s of both mononuclear and  i s c o n f i n e d mainly to the g r a f t bed  37  the  5a).  infiltration  a peak a t a d e n s i t y o n l y s l i g h t l y  (Figure 5b).  and  after transplantation  i n the g r a f t d e r m i s ' i n d i c a t e s  i s not h y p e r p l a s t i c or n e c r o t i c ( F i g u r e  to reach  after transplantation.  h i s t o l o g i c a l f e a t u r e s of i s o g r a f t h e a l i n g are sum-  marks the f o r m a t i o n  On  though  i s o g r a f t s are e s s e n t i a l l y i n d i s t i n g u i s h a b l e from  at the edges of the i s o g r a f t by  but  Epi-.  four poly-  and  lower  38  TABLE I .  Days s i n c e transplantation  H i s t o l o g i c a l f e a t u r e s of s k i n i s o g r a f t h e a l i n g .  Epithelium may be s e p a r a t e d from dermis; no h y p e r p l a s i a ;  Cellular  infiltration  v e r y m i l d ; m o s t l y polymorphonuclear  no n e c r o s i s  10  h y p e r p l a s t i c n e a r edge of g r a f t  mild; greater proportion of mononuclear c e l l s than at day 4  appearance normal e x c e p t f o r some h y p e r p l a s i a a t edge o f g r a f t  very mild; mostly p o l y morphonuclear  appearance normal  little  o r none  39  F i g u r e 5.  Skin  isografts  a.  4-day g r a f t  x200.  b.  6-day g r a f t x200. Only v e r y m i l d h y p e r p l a s i a and a l i g h t c e l l u l a r i n f i l t r a t i o n i n the g r a f t bed d i s t i n g u i s h e s skin i s o g r a f t s from normal s k i n . There a r e no s i g n s o f e p i t h e l i a l n e c r o s i s a t any s t a g e .  41  p a r t o f the dermis. ate  Near the edges o f t h e g r a f t  i t may be somewhat  hyperplastic,  n e c r o s i s never occurs. By  the  does n o t s e p a r -  from the dermis d u r i n g h i s t o l o g i c a l p r e p a r a t i o n and i s normal i n  appearance. but  The e p i d e r m i s a t t h i s s t a g e u s u a l l y  the e i g h t h day, only s c a t t e r e d  polymorphonuclear c e l l s and  o c c a s i o n a l mononuclear c e l l remain i n the g r a f t , and the e p i d e r m i s  looks e n t i r e l y normal.  Only the d e n s i t y o f f i b r o b l a s t s  lation  tissue  itself  from the s u r r o u n d i n g h o s t t i s s u e .  i n the granu-  of t h e g r a f t bed enables one t o d i s t i n g u i s h  i s even more d i f f i c u l t  to d i s t i n g u i s h  the g r a f t  By the t e n t h day, the g r a f t  from normal s k i n ,  and i s a p p a r e n t l y  permanently h e a l e d i n .  B.  H i s t o l o g i c a l Features of Skin A l l o g r a f t  Rejection  1) G e n e r a l O b s e r v a t i o n s The  process of a l l o g r a f t r e j e c t i o n  the h e a l i n g - i n  o c c u r s i n t h r e e major phases:  phase, i n which g r a n u l a t i o n t i s s u e  t i s s u e becomes f i r m l y  forms and t h e g r a f t e d  a t t a c h e d t o t h e h o s t ; the acute r e j e c t i o n  i n which the g r a f t  i s a t t a c k e d and k i l l e d ;  i n which the g r a f t  i s s l o u g h e d and the wound r e p a i r e d  new t i s s u e  a)  and the replacement phase, w i t h a growth o f  from the h o s t .  The h e a l i n g - i n For  retain  the f i r s t  phase few days a f t e r t r a n s p l a n t a t i o n ,  allografts  the appearance o f normal s k i n and a r e i n d i s t i n g u i s h a b l e  isografts.  phase,  from  The e p i d e r m i s may show m i l d h y p e r p l a s i a , and a s l i g h t  42  i n f i l t r a t i o n o f mononuclear c e l l s may o c c u r i n the g r a f t bed ( F i g u r e 6a).  Marked e p i d e r m a l h y p e r p l a s i a occurs o n l y a t the edge o f the g r a f t  (Figure 6b).  A p r o l i f e r a t i o n of f i b r o b l a s t s  i n the g r a f t bed and a t  the  edges o f the g r a f t marks the f o r m a t i o n o f g r a n u l a t i o n t i s s u e and  the  repairing  o f the wound  ( F i g u r e 6 c ) . The p r o l i f e r a t i o n o f s m a l l  b l o o d v e s s e l s i s apparent i n the g r a f t bed by t h e f o u r t h i n v a d e s the lower l a y e r s day  o f the g r a f t  O f t e n as e a r l y and  dermis ( F i g u r e 6 d ) .  o r s i x t h day, the g r a f t appears f i r m l y  b) The acute r e j e c t i o n  almost i n v a r i a b l y  day, and  quickly  By the f i f t h  a t t a c h e d t o the h o s t .  phase  as the f o u r t h  by t h e f i f t h  day a f t e r  or s i x t h  transplantation,  day, even b e f o r e the h e a l i n g  p r o c e s s i s complete, a l l o g r a f t s b e g i n t o show d e g e n e r a t i v e changes which do n o t o c c u r i n i s o g r a f t s .  The m i l d l y  hyperplastic  epidermis  takes on a somewhat d i s o r g a n i z e d appearance and the b a s a l c e l l s and  s e p a r a t e from each o t h e r ( F i g u r e 7 ) .  shrink  On the s i x t h day, the mono-  n u c l e a r c e l l i n f i l t r a t i o n o f the g r a f t bed i s markedly g r e a t e r than on the  fourth  day, and i s b e g i n n i n g to p e n e t r a t e the dermis o f the g r a f t  (Figure 7).  The b l o o d v e s s e l s o f the g r a f t bed a r e f r e q u e n t l y  w i t h mononuclear c e l l s perhaps about t o m i g r a t e out i n t o (as  hyperplastic  are  the seventh day, the e n t i r e  g r a f t e p i d e r m i s may be markedly  ( F i g u r e s 8, 9) and s c a t t e r e d  the s u r f a c e and i n the h a i r f o l l i c l e s . n o r m a l l y somewhat b a s o p h i l i c ,  even f a i n t l y e o s i n o p h i l i c . and  the t i s s u e s  i n Figure 6a). By  at  lined  necrotic  Cellular infiltration days.  can be found  The e p i d e r m a l c e l l s , which  now s t a i n l e s s  more e x t e n s i v e than on p r e v i o u s  cells  d e n s e l y , and some a r e o f the g r a f t  i s denser  43  F i g u r e 6.  S k i n a l l o g r a f t s i n the h e a l i n g - i n phase.  3-day g r a f t x200. The e p i d e r m i s i s t h i n and d e n s e l y - s t a i n i n g , and c e l l u l a r i n f i l t r a t i o n of the g r a f t bed i s l i g h t . e , d pa pc  epidermis , . graft dermis p a n n i c u l u s adiposus . , p a n n i c u l u s carnosus  _ , , g r a f t bed £j  3-day g r a f t x400. Some e p i d e r m a l h y p e r p l a s i a i s apparent a t the edge of the g r a f t as t h e wound i s r e p a i r e d . e  epidermis  E p i d e r m a l h y p e r p l a s i a and the f o r m a t i o n of new c o n n e c t i v e t i s s u e (dermis) a t the edge o f a 4-day g r a f t x600. e ct  epidermis connective tissue  (dermis)  Marked p r o l i f e r a t i o n o f b l o o d v e s s e l s i n the bed and o f a 4-day g r a f t x600. c  capillary  dermis  45  Figure 7.  Skin a l l o g r a f t s entering the acute rejection phase.  a.  4-day graft x400.  b.  4-day graft x200.  Epidermal hyperplasia i s moderate and c e l l u l a r i n f i l t r a t i o n of the graft bed i s l i g h t . Basal c e l l s i n the epidermis may show d i s t i n c t separation. Occasional pyknotic n u c l e i and vacuolated c e l l s can be found. Degree of g r a f t r e j e c t i o n : Epithelial  survival:  90%  2  46  [  47  F i g u r e 8.  S k i n a l l o g r a f t s undergoing  a.  5-day g r a f t  x200.  b.  6-day g r a f t  x200.  acute  rejection.  The e p i d e r m i s o f g r a f t s a t t h i s s t a g e shows more s e v e r e h y p e r p l a s i a than a t e a r l i e r s t a g e s ; o c c a s i o n a l c e l l s i n the h a i r f o l l i c l e s and sebaceous glands a r e p y k n o t i c or v a c u o l a t e d . The c e l l u l a r i n f i l t r a t e i s denser but s t i l l c o n f i n e d to the g r a f t bed and lower l a y e r s o f the dermis. Degree o f g r a f t  rejection:  Epithelial survival:  F i g u r e 9.  3  g r e a t e r than  50%  S k i n a l l o g r a f t s undergoing  a.  6-day g r a f t  x200.  b.  7-day g r a f t  x200.  acute  rejection.  The g r a f t e p i d e r m i s e x h i b i t s s e v e r e h y p e r p l a s i a w i t h many c e l l s cont a i n i n g v a c u o l e s or p y k n o t i c n u c l e i ( a r r o w s ) . H a i r f o l l i c l e s and sebaceous glands e x h i b i t w i d e s p r e a d n e c r o s i s . The dense mononuclear c e l l i n f i l t r a t e a t t h i s s t a g e extends w e l l i n t o the dermis and may even r e a c h the e p i d e r m i s i n some g r a f t s . Degree o f g r a f t  rejection:  Epithelial survival:  less  4 than  50%  49  By and  often  the e i g h t h  and n i n t h days, e p i t h e l i a l  i s widespread  the h a i r f o l l i c l e s and sebaceous glands a r e l o s i n g t h e i r  structure.  The e p i d e r m i s i s s e v e r e l y h y p e r p l a s t i c and may be  from t h e dermis by a dense l a y e r o f i n v a d i n g cells  necrosis  are f a i n t l y  cells.  Many  epithelial  e o s i n o p h i l i c ; these a r e o f t e n v a c u o l a t e d  contain n u c l e i that are e i t h e r pyknotic  or enlarged  C e l l u l a r i n f i l t r a t i o n o f the g r a f t i s very  separated  and may  and v e s i c u l a r .  dense, o f t e n e x t e n d i n g i n t o  the e p i d e r m i s i t s e l f . By  the tenth  day a f t e r t r a n s p l a n t a t i o n , many a l l o g r a f t s a r e  essentially  dead ( F i g u r e 10).  and  i s very  thin  and  f o l l i c u l a r s t r u c t u r e s may p e r s i s t i n some cases,  reveals  The e p i d e r m i s has l o s t  and e o s i n o p h i l i c .  A l t h o u g h the o u t l i n e o f e p i d e r m a l close  elements themselves have d i s i n t e g r a t e d .  s t i l l p r e d o m i n a n t l y mononuclear, the c e l l u l a r seems t o c o n t a i n leukocytes  a somewhat g r e a t e r p r o p o r t i o n  than on p r e v i o u s  Initial  o f polymorphonuclear  days.  changes i n the e p i d e r m a l c e l l s  of the mononuclear c e l l i n f i l t r a t e .  o f an a l l o g r a f t a r e  I n some c a s e s ,  changes a r e apparent.  appear p a l e infiltrate  Often,  necrosis  cells  o f the c e l l u l a r  t o the lower l a y e r s o f the dermis.  marked e p i t h e l i a l  cells  any marked de-  however, t h e e p i d e r m a l  and n e c r o t i c even though the g r e a t e r p a r t i s confined  or proximity  mononuclear  are seen i n the lower s t r a t a o f the e p i d e r m i s b e f o r e gradative  Though  i n f i l t r a t e i n the g r a f t  always o r even u s u a l l y a s s o c i a t e d w i t h the d e n s i t y  cases,  inspection  t h a t they a r e h e a v i l y i n f i l t r a t e d w i t h mononuclear c e l l s and  that the e p i t h e l i a l  not  i t s hyperplasia  i s a s s o c i a t e d w i t h only  I n some a very  light  50  Figure 10.  Skin a l l o g r a f t s i n the l a t e r part of the acute rejection phase.  a.  9-day graft x200.  b.  9-day graft x200.  These a l l o g r a f t s are e s s e n t i a l l y dead: h a i r f o l l i c l e s and sebaceous glands have l o s t most of t h e i r structure and are densely i n f i l t r a t e d with mononuclear and polymorphonuclear c e l l s . The epidermis, however, may s t i l l show severe hyperplasia and occasional n u c l e i retain t h e i r basophilia. Degree of graft r e j e c t i o n : Epithelial survival:  Figure 11.  5  less than 10%  Skin a l l o g r a f t s entering the replacement phase.  a.  10-day graft x200.  b.  12-day graft x200.  A l l epidermal elements i n these g r a f t s , and even large portions of the dermis, have degenerated. Polymorphonuclear c e l l s appear more abundant than i n grafts at e a r l i e r stages. Degree of graft r e j e c t i o n : Epithelial survival:  0%  6  51  52  cell infiltration  t h a t i s c o n f i n e d almost  entirely  to the g r a f t bed.  In o t h e r c a s e s , a h y p e r p l a s t i c b u t n o n - n e c r o t i c e p i t h e l i u m i s a s s o c i a t e d w i t h an e x c e p t i o n a l l y dense and e x t e n s i v e c e l l u l a r  c) The replacement By  infiltrate.  phase  the e l e v e n t h o r t w e l f t h day, v i r t u a l l y  the e n t i r e  graft  e p i d e r m i s i s dead and i s b e i n g s l o u g h e d a l o n g w i t h p a r t o f the g r a f t dermis  ( F i g u r e 11).  polymorphonuclear  The c e l l u l a r i n f i l t r a t e i s s t i l l  v e r y dense, b u t  l e u k o c y t e s , p a r t i c u l a r l y e o s i n o p h i l s , and macrophages  c o n t a i n i n g p h a g o c y t i z e d m a t e r i a l a r e more abundant than on e a r l i e r ( F i g u r e 11).  At the edges o f the g r a f t , the h o s t e p i d e r m i s has begun  to grow inwards, beneath t h a t has formed graft bed  dermis  days  the dead g r a f t  ( F i g u r e 12a).  and o v e r the  granulation tissue  I n many c a s e s , the deeper p a r t o f the  appears n o t t o be s l o u g h e d , b u t t o remain  i n the g r a f t  t o be c o v e r e d by t h i s new growth o f e p i t h e l i u m ( F i g u r e 12a).  By  the f o u r t e e n t h o r s i x t e e n t h day, h o s t e p i d e r m i s may cover much o f the former g r a f t s i t e ,  and the c e l l u l a r i n f i l t r a t e i s b e g i n n i n g t o d e c l i n e .  The new growth o f e p i d e r m i s appears  t o be i n v a d i n g the dermis  v a l s i n the f o r m a t i o n o f new h a i r f o l l i c l e s  2)  at i n t e r -  ( F i g u r e s 12b, 1 3 ) .  H i s t o l o g i c a l C r i t e r i a f o r A s s e s s i n g Degree o f A l l o g r a f t The h i s t o l o g i c a l changes o c c u r r i n g i n s k i n g r a f t s  r e j e c t i o n can be used as c r i t e r i a of an i n d i v i d u a l g r a f t .  Only  Survival  undergoing  f o r d e t e r m i n i n g the s t a g e o f r e j e c t i o n  those changes which o c c u r d u r i n g the  acute r e j e c t i o n phase, however, a r e o f r e l e v a n c e i n such a scheme. The  c o n d i t i o n o f the e p i d e r m i s a t the s u r f a c e o f the g r a f t and i n the  F i g u r e 12.  S k i n a l l o g r a f t s i n the replacement phase.  As the a l l o g r a f t i s sloughed o f f , the h o s t e p i d e r m i s grows out over the newly-formed dermis to r e p a i r the wound. The r e g i o n bounded by the lower r e c t a n g l e i s p i c t u r e d i n b below, and by the upper r e c t a n g l e i n F i g u r e 13.  16-day g r a f t x600. The new e p i d e r m i s appears to be f o r m a t i o n of new h a i r f o l l i c l e s  i n v a d i n g the dermis i n (arrows).  the  54  Old g r a f t bed A  55  F i g u r e 13.  S k i n a l l o g r a f t i n the replacement  phase.  The r e j e c t e d 16-day g r a f t i s s e p a r a t e d e n t i r e l y from the h o s t by a t h i n l a y e r of new e p i d e r m i s . The e p i d e r m i s appears t o be i n v a d i n g the dermis i n the f o r m a t i o n of new h a i r f o l l i c l e s ( a r r o w s ) ( x 6 0 0 ) .  56  57  hair f o l l i c l e s , philia,  as demonstrated by the degree o f h y p e r p l a s i a and baso-  and by the frequency  o f n e c r o t i c c e l l s , i s the major i n d i c a t o r  of the e x t e n t o f g r a f t s u r v i v a l .  The d e n s i t y and e x t e n t o f the c e l l u l a r  i n f i l t r a t e which accompanies g r a f t  r e j e c t i o n i s a l s o o f some h e l p i n  a s s e s s i n g the magnitude o f the r e a c t i o n . degrees o f g r a f t r e j e c t i o n c o r r e s p o n d i n g tages  Using  t o s u c c e s s i v e l y lower  o f s u r v i v i n g e p i t h e l i u m can be d e s c r i b e d Stage one d e s c r i b e s those  abnormality skin.  these c r i t e r i a , s i x percen-  (Table I I ) .  g r a f t s which show no r e a l s i g n s o f  and which a r e e s s e n t i a l l y  i n d i s t i n g u i s h a b l e from n o r m a l  Some very m i l d e p i d e r m a l h y p e r p l a s i a may be p r e s e n t , b u t no s i g n s  of n e c r o s i s can be found.  Few i f any mononuclear c e l l s have e n t e r e d  the g r a f t e d a r e a . Stage two ( F i g u r e 7) d e s c r i b e s those a l l o g r a f t s which show e a r l y but d e f i n i t e signs of abnormality. o r s e v e r e l y h y p e r p l a s t i c and t h e c e l l s some s e p a r a t i o n .  The e p i d e r m a l  The e p i d e r m i s  i s moderately  o f the b a s a l l a y e r may show  c e l l s s t a i n l e s s densely  than do the  c e l l s o f normal s k i n , and the c e l l membranes a r e w e l l - d e f i n e d . occasional pyknotic n u c l e i or vacuolated c e l l s l e a s t 90% o f t h e e p i d e r m i s  Stage t h r e e a l l o g r a f t s necrosis cells  than do s t a g e  can be found, b u t a t  appears h e a l t h y and v i a b l e .  at t h i s s t a g e shows o n l y a very  light  Very  infiltration  The g r a f t bed  o f mononuclear  cells.  ( F i g u r e 8) demonstrate somewhat more  two a l l o g r a f t s .  P y k n o t i c n u c l e i and v a c u o l a t e d  a r e more f r e q u e n t , h y p e r p l a s i a may be more s e v e r e , and the c e l l u l a r  i n f i l t r a t e i s denser appears  viable.  and more e x t e n s i v e .  A t l e a s t 50% o f the e p i d e r m i s  TABLE I I .  Figure  H i s t o l o g i c a l f e a t u r e s o f s i x stages o f s k i n a l l o g r a f t r e j e c t i o n i n normal mice.  Degree o f graft rejection  Time a f t e r transplantation  % epithelial survival  6  1  1-3  days  100%  7  2  4- 5 days  90%  5- 7 days  7-8  10  9-11  11  11+  days  days  days  Swelling and hyperplasia  Staining character  Necrosis  Cellular infiltration Mono Poly  mild  basophilic  none  +-  moderate  lightly basophilic  rare  +  >50%  severe  lightly basophilic  scattered  + or  <50%  severe  mixed basop h i l i c and eosinophilic  widespread  10%  severe  eosinophilic  widespread  II H  eosinophilic  complete  +++  0%  59  Stage f o u r with less  allografts  (Figure  9) show r a t h e r  than 50% o f the e p i t h e l i u m s u r v i v i n g .  s e v e r e damage,  Epidermis, h a i r  c l e s , and sebaceous glands r e t a i n t h e i r g e n e r a l s t r u c t u r e , is  widespread.  Many c e l l s  are e o s i n o p h i l i c ,  but necrosis  and some a r e v a c u o l a t e d ;  many c o n t a i n e i t h e r e n l a r g e d , v e s i c u l a r n u c l e i  or pyknotic n u c l e i .  E p i d e r m a l h y p e r p l a s i a i s s e v e r e , and t h e mononuclear c e l l may e x t e n d i n t o the e p i d e r m i s  folli-  infiltrate  itself.  A l l o g r a f t s a t s t a g e f i v e a r e e s s e n t i a l l y dead, w i t h l e s s 10%  surviving  epithelium  (Figure  10).  and  sebaceous glands have l o s t most o f t h e i r s t r u c t u r e ,  i n f i l t r a t e d w i t h mononuclear c e l l s . p l a s t i c and i s o f t e n vading c e l l s . scattered  I n many c a s e s , t h e h a i r  follicles  and a r e d e n s e l y  The e p i d e r m i s i s s e v e r e l y  hyper-  s e p a r a t e d from the dermis by a dense l a y e r o f i n -  Most o f t h e e p i d e r m a l c e l l s  nuclei  than  r e t a i n some b a s o p h i l i a .  are e o s i n o p h i l i c ,  although  The c e l l b o u n d a r i e s a r e o f t e n  distinct. A l l o g r a f t s a t s t a g e s i x a r e u n q u e s t i o n a b l y e n t i r e l y dead 11). and  The e p i d e r m i s has l o s t i t s h y p e r p l a s t i c eosinophilic;  individual cells  In some c a s e s , the o u t l i n e p e r s i s t , but close  is  disintegrating.  replacement phase.  to d i s t i n g u i s h  at a l l .  o f e p i d e r m a l and f o l l i c u l a r s t r u c t u r e s  inspection  elements a r e t o t a l l y  appearance, and i s t h i n  are d i f f i c u l t  reveals  that  they a r e h e a v i l y  w i t h mononuclear and p o l y m o r p h o n u c l e a r c e l l s . lial  (Figure  may  infiltrated  In other cases,  epithe-  degenerate and even the dermis o f t h e g r a f t  Allografts  a t t h i s s t a g e a r e about t o e n t e r the  60  3) MST o f DBA/2 S k i n A l l o g r a f t s The  distribution of graft  g r a f t s on C57B1/6 +/+ r e c i p i e n t s  on C57B1/6 +/+ Mice  r e j e c t i o n scores  f o r DBA/2 s k i n  i s shown i n T a b l e I I I .  allo-  Statistical  a n a l y s i s o f these d a t a u s i n g the s i m p l i f i e d method o f e v a l u a t i n g doseresponse e f f e c t s  d e s c r i b e d by L i t c h f i e l d and W i l c o x o n  t h a t t h e median s u r v i v a l time (MST) o f these 95%  confidence  intervals  C.  A l l o g r a f t R e j e c t i o n i n W/W  allograft  (1948) r e v e a l s i s 9.7 days.  f o r t h i s v a l u e a r e 8.6 days and 11.1 days.  V  Mice  1) On t h e EighthDay A f t e r T r a n s p l a n t a t i o n The  d i s t r i b u t i o n of graft  r e j e c t i o n scores  f o r DBA/2 s k i n  allo-  g r a f t s e x c i s e d on the e i g h t h day a f t e r t r a n s p l a n t a t i o n t o 18 +/+ mice, 25 W/+ mice, 13 W /+  mice, and 34 W/W  V  V  mice i s summarized i n T a b l e IV.  S t a t i s t i c a l a n a l y s i s u s i n g t h e Mann-Whitney U-Test t h a t +/+ s c o r e s a r e n o t s i g n i f i c a n t l y +/+, W/+,  and W /+ V  single population.  different  ( S i e g e l , 1956) shows  from W/+  and W /+  scores;  V  d a t a can t h e r e f o r e be t r e a t e d as samples from a Comparison of W/W  V  s c o r e s w i t h +/+, W/+,  and W /+ V  s c o r e s , a g a i n u s i n g the Mann-Whitney U-Test, i n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e between t h e two samples  (p = .0036); the two groups can n o t  t h e r e f o r e be c o n s i d e r e d t o be drawn from the same p o p u l a t i o n .  2) MST o f DBA/2 S k i n A l l o g r a f t s on +/+, W/+, W /+, V  and W/W  V  M o r t a l i t y data f o r DBA/2 s k i n a l l o g r a f t s on 45 W/W  V  52 o f t h e i r +/+, W/+,  and W /+  These data were a n a l y z e d  V  Hosts  mice and  l i t t e r m a t e s a r e summarized i n T a b l e V.  u s i n g t h e s i m p l i f i e d method o f e v a l u a t i n g  61  TABLE I I I .  Graft r e j e c t i o n scores determined by h i s t o l o g i c a l analysis of DBA/2 skin a l l o g r a f t s on C57B1/6 +/+ hosts.  Days a f t e r transplantation  3  a Graft  rejection  1 1 1 2  4  2 2 3 1 2 1  5  112  6  12 2 12 1  7  3 3 3 3 2 1  8 9 10  2 11  5 5 4 4 2 2 4 3 5 5 4 5 4 3 4 6 4 6 4 6 5 4 5  11  5 5 4  12  5 5 5 5 5 5  13  5 6 6 5 5 6  14  6 6 6 6 6 6  •k  scores  Each number i n this column represents a single a l l o g r a f t on a single mouse.  TABLE IV.  D i s t r i b u t i o n of graft r e j e c t i o n scores of DBA/2 skin a l l o g r a f t s on +/+, W/+, W /+, and W/W mice; grafts were excised on the eighth day a f t e r transplantation. v  v  Number of mice obtaining Graft r e j e c t i o n score  each score  +/+  W/+  w /+  w/w  1  3  4  1  3  2  1  10  2  3  3  5  6  6  7  4  6  3  2  10  3  1  2  9  0  1  0  2  n =  18  25  13  34  % mortality =  16.8  15.4  32.3  *  5  * 6  8.0  v  v  Graft r e j e c t i o n scores of 5 or 6 indicate that the graft i s dead.  63  TABLE V.  M o r t a l i t y data f o r DBA/2 s k i n a l l o g r a f t s on +/+, W / + , W / + , and W / W h o s t s . v  Days s i n c e transplantation  v  Mortality +/+, W/+, w / +  w / w  v  # dead # sampled  % mortality  # dead # sampled  0  0/13  8  1/8  13  3/12  25  9  3/11  27  3/7  43  10  10/16  63  9/14  64  11  4/4  100  3/3  100  12  1/1  100  95%  9.7 days  0/9  % mortality  7  MST  0  v  9.3 days  confidence  l i m i t s o f MST 9.3-10.3 days  8.4-10.2 days  64  dose-effect experiments described by L i t c h f i e l d and Wilcoxon (1949). Dose-effect straight l i n e s f o r each sample were plotted on logarithmicp r o b a b i l i t y paper. and W/+ V  The L D  5 0  or MST of DBA/2 a l l o g r a f t s on +/+, W/+,  hosts given by this method i s 9.7 days; 95% confidence l i m i t s  for this value are 9.3 days and 10.3 days. on W/W  V  The MST of DBA/2 grafts  hosts as read from the graph i s 9.3 days; 95% confidence l i m i t s  for this value are 8.4 days and 10.2 days. Comparison of the two l i n e s indicates that they do not deviate s i g n i f i c a n t l y from p a r a l l e l i s m .  Thus, the mortality rates of the two  samples are not s i g n i f i c a n t l y d i f f e r e n t .  A comparison of the MST values  of the two samples indicates that these parameters are not s i g n i f i c a n t l y different. 3) Grafts Exchanged Across Weak Histocompatibility Barriers The graft r e j e c t i o n scores of skin from male +/+ mice transplanted to female +/+ and W/W  V  hosts are shown i n Table VI.  The MST  of a l l o g r a f t s on +/+ hosts would appear to be about 20 or 22 days; u n t i l day 20, more than 50% of the transplants on +/+ hosts are s t i l l a l i v e (scoring less than 5).  Although the number of W/W  hosts i n  V  this experiment i s too small to allow d e f i n i t e conclusions to be drawn, v i t i s i n t e r e s t i n g to note that none of the grafts to W/W  mice showed  any surviving epithelium, thus strongly suggesting a shorter MST f o r grafts on these mice.  65  TABLE V I .  G r a f t r e j e c t i o n s c o r e s f o r g r a f t s o f s k i n from +/+ male donors t r a n s p l a n t e d t o +/+ and W/W female h o s t s . v  Graft  Days a f t e r transplantation  +/+.  * 3,3,4,5  17  2,2,2,3,3 * * * 4,5 ,6 ,6 * 3,4,6 * * * * 4,5 ,6 ,6 ,6  18 20 22 24  Graft is  dead.  r e j e c t i o n scores  rejection  w/w  v  * 5 * * 5 ,5  * 6 * * 6 ,6  scores of 5 or 6 i n d i c a t e  that  the g r a f t  66 II.  A.  LYMPHOCYTE ACTIVATION STUDIES  L a b e l l i n g in v i v o The  uptake o f t r i t i a t e d  b l o o d o f +/+ and W/W  V  thymidine  by c e l l s o f the p e r i p h e r a l  mice b e a r i n g no g r a f t s o r b e a r i n g s k i n  i s shown i n T a b l e V I I .  Results are expressed  allografts  as d i s i n t e g r a t i o n s p e r  minute p e r c u l t u r e (.05 ml w h i t e b l o o d ) and as d i s i n t e g r a t i o n s p e r minute p e r 1 0  5  white blood c e l l s  The mean DPM/10 lower  than  5  i n each  culture.  w h i t e b l o o d c e l l s o f u n g r a f t e d +/+ mice i s  t h a t o f +/+ mice b e a r i n g a l l o g r a f t s .  of two u n g r a f t e d W/W  V  U n f o r t u n a t e l y , samples  mice were l o s t , and comparisons between g r a f t e d  and u n g r a f t e d mutant mice can n o t be made. S t a t i s t i c a l a n a l y s i s u s i n g an u n p a i r e d t - t e s t i n d i c a t e s t h a t on the e i g h t h day a f t e r t r a n s p l a n t a t i o n , mean DPM/10  5  white blood  from +/+ mice a r e s i g n i f i c a n t l y d i f f e r e n t from mean DPM/10 cells  from W/W  v  mice.  5  cells  white blood  Means a t day seven, however, a r e n o t s i g n i f i c a n t l y  d i f f e r e n t , and on days n i n e and t e n the samples a r e t o o s m a l l f o r meaningf u l comparisons to be made. mean DPM/10  5  white blood c e l l s  from mean DPM/10  B.  I f a l l days a r e taken t o g e t h e r , however,  5  from +/+ mice a r e s i g n i f i c a n t l y  white blood c e l l s  from W/W  V  different  mice.  Labelling i n vitro The  uptake o f t r i t i a t e d  b l o o d o f +/+ and W/W  v  thymidine  by c e l l s o f the p e r i p h e r a l  mice b e a r i n g s k i n a l l o g r a f t s  V I I I and summarized i n F i g u r e 14.  i s shown i n T a b l e  Results a r e expressed  as d i s i n t e -  g r a t i o n s p e r minute (DPM) p e r c u l t u r e (.1 ml whole b l o o d ) .  Since  only  67  TABLE V I I .  Days a f t e r transplantation  * 7  *  Uptake of t r i t i a t e d thymidine by c e l l s of the p e r i p h e r a l b l o o d o f mice b e a r i n g s k i n a l l o g r a f t s . L a b e l l i n g done i n v i v o .  5  WBC  +/+  w/w  +/+  w/w  1,395 1,319 1,110 1,072  1,639 1,051  698 222 291 509  815 629  1,092 909  8  DPM/10  DPM/culture  1,121 1,012 1,139  v  1,562  360 266  1,020 1,198 1,391 2,613 1,036  436 256 397  v  577 455 774 384 409 516  9  1,125  1,108  534  820  10  1,159 1,169  1,143  731 322  543  223 155 261 142 258 127 108 158  1,165 986 1,082 913 969 249 356 462  no graft  *  Differences between +/+ and Student's t - t e s t , a = .025.  W/W mice are s i g n i f i c a n t by V  68  TABLE V I I I .  Days a f t e r transplantation 4  Uptake o f t r i t i a t e d thymidine by c e l l s o f the p e r i p h e r a l b l o o d o f mice b e a r i n g s k i n a l l o g r a f t s . L a b e l l i n g done i n v i t r o .  DPM / c u l t u r e +/+  w/w  674 1,148 673  877 1,785 514  500 524 502  1,400 1,114 1,104 917  8  14  Day 8 a u t o g r a f t s  No g r a f t s  v  289 730 414  1,201 1,109 1,615  62 157  95 370  451 713  171  Mean No. o f lymphocytes/culture +/+ W/W  DPM/10 lymphocytes +/+ W/W  367,700  183.3 312.2 183.0  294.1 578.6 172.4  163.6 171.5 164.3  458.6 364.9 361.6 300.4  57.1 144.2 81.8  271.5 250.7 365.1  V  305,600  298,200  5  305,300  434,700  595,800.  14.3 31.0  15.9 62.1  665,700  69.7 110.9  26.8  646,600  Minus background and DPMs " i n c o r p o r a t e d " a t z e r o time (- 600 DPM).  <n  500  -H  o  400  sz  CL  H  300  9  200  Q.  allografts  +/ +  100  DL  Q  Figure  14.  ?  w/w" ~ "•  0  4  autografts  v  Days a f t e r  8  14  Transplantation  Uptake of t r i t i a t e d thymidine by c e l l s of the p e r i p h e r a l b l o o d of mice bearing s k i n a l l o g r a f t s or s k i n autografts. L a b e l l i n g done i n v i t r o .  ON VO  70  a c t i v a t e d lymphocytes a r e known to i n c o r p o r a t e t r i t i a t e d t h y m i d i n e , however, r e s u l t s a r e a l s o e x p r e s s e d as DPM p e r 1 0  5  lymphocytes.  Since  the number o f lymphocytes may v a r y between c u l t u r e s , t h e c o n v e r s i o n o f DPM p e r c u l t u r e t o DPM p e r number o f lymphocytes p r e s e n t i s a more satisfactory  r e p r e s e n t a t i o n o f the r e s u l t s and p r o v i d e s a more a c c u r a t e  b a s i s f o r comparison o f c o n t r o l and e x p e r i m e n t a l groups. DPM/10  5  lymphocytes  from +/+ and W/W  or b e a r i n g a u t o g r a f t s are c l e a r l y +/+ and W/W  V  mice b e a r i n g no g r a f t s  V  lower than DPM/10  mice b e a r i n g a l l o g r a f t s .  a comparison between +/+ mice and W/W  V  5  lymphocytes  The sample i s too s m a l l t o a l l o w mice b e a r i n g no g r a f t s , b u t among  those b e a r i n g a u t o g r a f t s a n a l y s i s w i t h an u n p a i r e d t - t e s t no  indicates  difference. F u r t h e r s t a t i s t i c a l a n a l y s i s w i t h an u n p a i r e d t - t e s t  t h a t on the f o u r t h day a f t e r t r a n s p l a n t a t i o n , mean DPM/10 from +/+ mice a r e n o t d i f f e r e n t W/W  V  from mean DPM/10  5  indicates  lymphocytes  lymphocytes  5  mice; however, on the e i g h t h day and f o u r t e e n t h day a f t e r  p l a n t a t i o n , mean DPM/10 lower than mean DPM/10  5  5  III.  A.  from  lymphocytes lymphocytes  from trans-  from +/+ mice a r e s i g n i f i c a n t l y from W/W  V  mice  ( a = .05).  HUMORAL IMMUNE RESPONSES  P r o d u c t i o n o f SRBC A g g l u t i n i n s i n +/+and W/W  Mice  V  Serum t i t e r s o f SRBC a g g l u t i n i n s produced by +/+ and W/W  V  mice  over a p e r i o d o f t e n weeks a r e shown i n T a b l e IX, and a r e summarized i n F i g u r e 15.  The p r i m a r y responses o f +/+ and W/W  V  mice- a r e e s s e n t i a l l y  TABLE IX.  Serum a g g l u t i n i n  titers  i n mice o f d i f f e r e n t genotype a f t e r immunization w i t h SRBC.  Serum a g g l u t i n i n Mouse #  Genotype  Days -> 0 4 0  100  0  200  0  300  0  300  0  400  1  +/+  2  W/W  3  +/+  4  w/w  5  +/+  6  W/W  7  +/+  8  w/w  9  +/+  10  w/w  v  v  v  v  v  6  titers  a f t e r immunization w i t h SRBC  20  24  8  10  14  400  800  400  400  800  600  400  400  400  200  200  600  400  400  800  400  600  600  800  400  400  600  800  1,600  800  1,600  800  400  800  800  400  600  0  400  600  800  0  200  400  400  28 3,200  32  36  4,800  6,400  1,200  1,600  42 6,400  52  72  3,200  2,400  800  300  1,200  3,200  4,800  6,400  6,400  4,800  400  1,200  3,200  2,400  3,200  2,400  1,200  600  800  3,200  2,400  4,800  4,800  1,600  1,200  400  1,600  1,600  3,200  4,800  3,200  800  300  1,600  800  3,200  3,200  1,200  800  800  3,200  4,800  1,200  800  20  200  300  400  800  400  600  600  1,600  0  150  300  200  400  200  400  600  1,600  1,600  3,200  3,200  1,600  800  0  200  300  400  800  400  400  400  400  400  800  800  800  800  I  10 15.  I  I  I  I  I  I  I  f 20 3 0 4 0 5 0 6 0 70 80 Ag Days a f t e r Transplantation  Serum a g g l u t i n i n t i t e r s of mice of d i f f e r e n t genotype a f t e r immunization with SRBC.  identical.  Most W/W  v  mice, l i k e +/+ mice, are c l e a r l y  capable o f p r o -  ducing a p r o l o n g e d secondary response to SRBC a n t i g e n s but t h i s  response  from the 32nd day onward, i s , on the average, s i g n i f i c a n t l y weaker than the secondary response produced by +/+ mice a = .05).  (Student's t - t e s t ,  I n some c a s e s , the secondary response o f W/W  a p p a r e n t l y normal, as i n mouse #6 and mouse #8.  mice may be  I n o t h e r c a s e s , the  response i s somewhat l e s s than normal, as i n mouse #2 and perhaps mouse #4.  A secondary response seems to be l a c k i n g e n t i r e l y  i n mouse #10,  where a g g l u t i n i n t i t e r s n e v e r exceeded  those o f the primary response.  B.  i n +/+, W/+,  P r o d u c t i o n of P l a q u e - f o r m i n g C e l l s  The numbers o f p l a q u e - f o r m i n g c e l l s +/+, W/+,  W /+, V  and W/W  V  genotypes  SRBC a r e shown i n T a b l e XA.  and W/W  V  Mice  (PFC) produced by mice o f  Each number i n the body o f the t a b l e from a s i n g l e mouse.  Spleens  mice produce 1/4 the number o f h e m o l y s i n - f o r m i n g c e l l s as  V  do s p l e e n s from +/+ mice. W/+  V  f o u r days a f t e r immunization w i t h  r e p r e s e n t s the mean o f t r i p l i c a t e samples from W/W  W /+,  and W /+ V  +/+ mice.  The number o f PFC produced by s p l e e n s from  mice i s i n t e r m e d i a t e between t h a t produced by W/W  V  and  A n a l y s i s o f t h e s e d a t a u s i n g the K r u s k a l l - W a l l i s One-Way  A n a l y s i s o f V a r i a n c e ( S i e g e l , 1956) i n d i c a t e s are drawn from d i f f e r e n t  t h a t the f o u r  samples  populations.  In normal mice, t h e peak PFC response t o SRBC a d m i n i s t e r e d intraperitoneally  o c c u r s a t f o u r days  (Wortis e_t a l . , 1966).  At t h i s  v time, as d i s c u s s e d above,  s p l e e n s o f W/W  fewer PFC than do s p l e e n s o f normal mice.  mice  contain  significantly  T e s t s were, however, a l s o  74  TABLE XA.  Numbers o f p l a q u e - f o r m i n g c e l l s produced by s p l e e n s o f mice o f d i f f e r e n t genotype f o u r days a f t e r exposure to SRBC.  # PFC/10  5  Spleen C e l l s  w/w  +/+  W/+  w /+  1  252  136  128  85  2  208  250  150  26  3  200  151  143  32  4  214  127  142  72  218  166  140  54  Trial #  Mean  TABLE XB.  Trial #  1 2  v  v  Numbers of p l a q u e - f o r m i n g c e l l s produced by s p l e e n s o f mice o f d i f f e r e n t genotype s i x days a f t e r exposure t o SRBC.  # PFC/10  5  Spleen  Cells  +/+  w/w  220  43  no d a t a  v  no data  75  performed on possibility be  the  s i x t h day  a f t e r immunization i n c o n s i d e r a t i o n  t h a t the k i n e t i c s o f the PFC  response i n W/W  V  a l t e r e d , w i t h the peak o c c u r r i n g somewhat l a t e r .  data are p r e s e n t e d i n T a b l e XB. PFC  response o f W/W  mice i s any h i g h e r  V  than a t  The V  mice might available  i n d i c a t i o n t h a t the peak  s i x days a f t e r immunization  MITOGEN STIMULATION STUDIES  uptake of t r i t i a t e d thymidine by  mice c u l t u r e d w i t h v a r i o u s  (LPS)  the  four.  IV.  W/W  There i s no  The  of  spleen  concentrations  or C o n c a n a v a l i n - A (Con-A), and  harvested  of  cells  o f +/+  and  lipopolysaccharide  a f t e r two,  three,  or  f o u r days o f c u l t u r e i s e x p r e s s e d as mean counts per minute (CPM), i s shown i n T a b l e XI. the mean CPM  Each number i n the body of the t a b l e  of quadruplicate  U s i n g these data,  c u l t u r e s of c e l l s  f o r each treatment and Both raw spleen  cells  cells,  regardless  t r e a t e d w i t h LPS of the  XII.  the mean s t i m u l a t i o n i n d i c e s of  concentration  of mitogen used or the  S t a t i s t i c a l a n a l y s i s of the  and W/W  V  CPM  are always lower than those of +/+  s t i m u l a t i o n i n d i c e s using  c o n f i r m s t h a t +/+  from each of s i x mice.  of RPMI c u l t u r e s ) were c a l c u l a t e d  a r e shown i n T a b l e  count means and  o f time i n c u l t u r e . of the  represents  s t i m u l a t i o n i n d i c e s ( s t i m u l a t i o n i n d e x = mean  o f c u l t u r e s w i t h mitogens/mean CPM  and  raw  samples are  (Siegel,  drawn from d i f f e r e n t  V  spleen  length  count means  the Mann-Whitney U - t e s t  W/W  and 1956)  populations.  76  TABLE X I .  Uptake o f t r i t i a t e d thymidine ( e x p r e s s e d as mean CPM) by s p l e e n c e l l s o f +/+ and w/wv mice c u l t u r e d w i t h v a r i o u s c o n c e n t r a t i o n s o f LPS and Con-A.  96 hour  72 hour  Mitogen concentration  48 hour  +/+  w/w  RPMI  2,430  2,782  v  w/w  +/+  w/w  907  1,337  1,071  1,834  +/+  v  v  LPS  5  44,285  40,430  23,514  13,948  11,493  2,166  LPS  20  47,721  37,165  27,677  16,162  13,453  2,891  LPS  40  42,374  38,919  19,758  16,167  8,717  8,371  Con-A  25  62,980  58,740  42,368  65,650  49,127  90,186  Con-A  . 50  63,210  62,545  47,001  66,610  59,455  76,162  Con-A 1. 00  42,900  40,595  33,388  40,975  41,491  72,721  TABLE X I I .  S t i m u l a t i o n i n d i c e s o f s p l e e n c e l l s from +/+ and W/W mice c u l t u r e d w i t h v a r i o u s c o n c e n t r a t i o n s o f LPS and Con-A, and l a b e l l e d w i t h t r i t i a t e d thymidine v  . +/+  96 hour  72 hour  48 hour  Mitogen concentration  w/w  v  +/+  w/w  +/+  v  w/w  v  LPS  5  18.2  14.5  25.9  10.4  10.7  1.2  LPS  20  19.6  13.4  30.5  12.1  12.6  1.6  LPS  40  17.4  14.0  21.8  12.1  8.1  4.6  Con-A  .25  25.9  21.1  46.7  49.1  45.9  49.2  Con-A  .50  26.0  22.5  51.8  49.8  55.5  41.5  Con-A 1.00  17.7  14.6  36.8  30.7  38.7  39.7  mean CPM o f c u l t u r e s w i t h mitogens mean CPM o f RPMI c u l t u r e s  78 v  S t i m u l a t i o n i n d i c e s of W/W are a p p r o x i m a t e l y  spleen c e l l s  t r e a t e d w i t h Con-A  e q u a l to s t i m u l a t i o n i n d i c e s of +/+  spleen  cells  t r e a t e d w i t h Con-A, r e g a r d l e s s of the c o n c e n t r a t i o n of mitogen used o r the l e n g t h of time i n c u l t u r e . Whitney U - t e s t c o n f i r m s same p o p u l a t i o n .  t h a t +/+  The mean raw  Statistical and W/W  samples are drawn from the  V  counts  a n a l y s i s u s i n g the Mann-  of W/W  spleen c e l l s  V  w i t h Con-A, however, appear t o be h i g h e r than those of +/+  treated spleen  cells  a f t e r 72 or 96 hours o f c u l t u r e , and s t a t i s t i c a l a n a l y s i s s u g g e s t s v t h a t +/+  and W/W  samples a t 96 hours are drawn from d i f f e r e n t  popu-  lations .  V.  A.  OTHER STUDIES  Macrophage M i g r a t i o n S t u d i e s The  capillary meter. of  a r e a covered by p e r i t o n e a l exudate c e l l s m i g r a t i n g  tubes  The  i n Mackaness-type chambers was  r e s u l t s a r e shown i n T a b l e X I I I .  the t a b l e r e p r e s e n t s  from  measured w i t h a p r a n i Each number i n the body  the mean of d u p l i c a t e samples of c e l l s  from  a s i n g l e mouse. The  a r e a c o v e r e d by m i g r a t i n g c e l l s v a r i e s markedly between  mice, r a n g i n g from 17.0 mean of 18.7 V  mm , 2  and  t o 22.5  mm  2  from 17.5  for cells  to 31.5  mm  from +/+  for cells  2  mice, w i t h from W/+  a  and  o  i  W /+ mice, w i t h a mean of 26.0  and  23.3  mnr  respectively.  For  cells  from W/W  mice, m i g r a t i o n o c c u r r e d over an a r e a r a n g i n g from 11.5  42.5  with  V  mm , 2  a mean of 21.7  mm . 2  There i s no  to  apparent d i f f e r e n c e  79 between the f o u r groups o f mice; s t a t i s t i c a l a n a l y s i s w i t h the K r u s k a l l W a l l i s One-way A n a l y s i s of V a r i a n c e ( S i e g e l , 1956) f o u r samples  confirms that  the  are drawn from the same p o p u l a t i o n .  TABLE X I I I .  A r e a (mm ) c o v e r e d by p e r i t o n e a l c e l l s from mice of d i f f e r e n t genotype a f t e r 8 hours o f m i g r a t i o n from c a p i l l a r y tubes i n Mackanesstype chambers. 2  +/+  W/+  W /+  W/W  22.0  21.5  29.5  26.5  13.0  25.0  17.5  22.5  18.0  31.5  36.0  13.0  21.5  12.5  27.0  16.5  21.0  18.0  V  V  22.5  18.0  17.0  11.5 25.5 12.5 42.5  B.  23.3  26.0  18.7  Lymphocyte Adhesion S t u d i e s The number o f lymph node c e l l s  a f t e r 45 minutes number o f c e l l s of  21.7  o f i n c u b a t i o n i s e x p r e s s e d as a p e r c e n t a g e o f the originally  lymph node c e l l s  T a b l e XIV.  adhering to glass cover s l i p s  r e s t i n g on the c o v e r s l i p .  from seven +/+  and seven W/W  V  mice  Results of  tests  are shown i n  Each number i n the body o f the t a b l e r e p r e s e n t s the mean  80  percentage  of c e l l s  adhering to cover s l i p s  chambers c o n t a i n i n g lymph node c e l l s no apparent  i n two d i f f e r e n t  from a s i n g l e mouse.  d i f f e r e n c e between the groups;  culture  There i s  a n a l y s i s w i t h t h e Mann-  Whitney U - t e s t confirms t h a t the two samples a r e drawn from t h e same population.  TABLE XIV.  P e r c e n t a g e o f lymph node c e l l s a d h e r i n g t o g l a s s cover s l i p s a f t e r 45 minutes i n c u b a t i o n .  P e r c e n t Adherence  Mean  +/+  28 30 33 40 46 46 52 .  39.3%  w/w  33 33 34 38 53 57 57  43.5%  Genotype  v  VI.  A.  HAEMATOLOGY  U n g r a f t e d Mice H a e m a t o l o g i c a l d a t a f o r u n g r a f t e d mice, 12 t o 16 weeks o l d ,  are shown i n T a b l e XV.  Mean w h i t e b l o o d c e l l counts  are c o n s i s t e n t l y lower than mean c e l l counts  f o r female mice  f o r u n g r a f t e d males, b u t  the d i f f e r e n c e s a r e n e i t h e r l a r g e n o r s t a t i s t i c a l l y s i g n i f i c a n t . variation exists f e r e n t genotypes,  i n t o t a l and d i f f e r e n t i a l  Some  counts between mice o f d i f -  b u t a g a i n the d i f f e r e n c e s a r e n o t s i g n f i c a n t .  81 TABLE XV.  Genotype  n  % Lymphocytes  T o t a l WBC/mm  3  +/+  9  7,356 ± 2,227 7,944 ± 2,347  87.9 ± 83.8 ±  5.8 10.6  10 11  W/+  9  5  5,936 ± 1,319 7,525 ± 3,048  80.3 ± 71.3 ±  8.3 18.6  12 16  w /+  9  5  6,377 ± 1,954 7,560 ± 2,787  88.5 ± 84.7 ±  4.0 9.0  15 8  w/w  9  6,727 ± 3,336 6,880 ± 3,36 7  94.9 ± 87.5 ±  1.2 10.0  10 11  cr  v  B.  T o t a l and d i f f e r e n t i a l w h i t e b l o o d c e l l counts of u n g r a f t e d +/+, W/+, W /+, and W/W mice.  v  5  Mice B e a r i n g S k i n A u t o g r a f t s o r S k i n  Allografts  Mice b e a r i n g s k i n a u t o g r a f t s o f e i g h t days s t a n d i n g appear t o have somewhat lower t o t a l w h i t e c e l l  counts than do u n g r a f t e d mice  ( T a b l e X V I ) , but the number o f mice sampled h e r e i s too s m a l l t o p e r m i t m e a n i n g f u l comparisons  to be made.  T o t a l w h i t e b l o o d c e l l counts i n mice b e a r i n g s k i n are  approximately h a l f  are  normal i n the e a r l y  six), the in  b u t i n l a t e r days  those o f u n g r a f t e d mice.  counts  ( t h a t i s , t o day  (days seven t o ten) t h e s e appear somewhat a l t e r e d , d e c r e a s i n g by 10 t o 15%.  Variability  counts o f these g r a f t e d mice i s g r e a t e r than t h a t i n  d i f f e r e n t i a l counts of u n g r a f t e d mice. in  Differential  days a f t e r t r a n s p l a n t a t i o n  mean p e r c e n t a g e o f lymphocytes differential  allografts  There appear t o be no  differences  any o f these parameters between mice o f d i f f e r e n t genotypes b e a r i n g  allografts.  TABLE XVI.  T o t a l and d i f f e r e n t i a l white blood c e l l counts of +/+, W/+, W/+, and V W/W mice bearing skin autografts or skin a l l o g r a f t s . V  w/w  +/+, W/+, w /+ v  Days a f t e r transplantation  v  n  Total WBC/mm  % lymphocytes  n  Total WBC/mm  % lymphocytes  to day 6  10  4,562 ± 1,530  80.6 ± 10.5  10  3,823 ± 2,012  78.0 ± 11.4  days 7 to 10  31  4,710 ± 1,822  64.7 ± 19.3  31  4,431 ± 1,530  68.9 ± 18.7  7  5,656 ± 2,035  5  4,424 ± 1,849  2  5,502  2  6,338  3  3  Allografts  day 14  No data  No data  Autografts day 8  79.0  94.0  DISCUSSION  The  investigations described i n this  the hope o f c l a r i f y i n g responses.  t h e s i s were undertaken i n  the e x t e n t to which the W l o c u s a f f e c t s  immune  No new i n f o r m a t i o n c o n c e r n i n g the e f f e c t s o f the W  i n mice has been p r e s e n t e d f o r some y e a r s , and the primary  mutation  s i t e of  a c t i o n o f t h i s gene has remained, u n t i l now, as much a mystery as e v e r . If  t h i s gene were found  to a f f e c t immune r e s p o n s e s , however, and i f  the n a t u r e o f t h i s e f f e c t  c o u l d be determined,  some new l i g h t  might  be shed on t h e mechanism o f a c t i o n o f t h i s e n i g m a t i c gene, and W mutant mice might thereby prove u s e f u l i n s t u d i e s o f immune r e a c t i o n s . the d i s c u s s i o n t o f o l l o w , the experiments  In  and d a t a t h a t have been  p r e s e n t e d h e r e w i l l be i n t e r p r e t e d i n terms o f what i s known about immune responses  i n g e n e r a l , and w i l l  then be examined f o r what they  r e v e a l about t h e a c t i o n o f the W l o c u s . my major experiments presented i n Table  c o n c e r n i n g the immune responses  o f W mice i s  XVII.  I.  A.  A summary o f the r e s u l t s o f  TRANSPLANTATION IMMUNITY  Genetics o f Transplantation 1) C h a r a c t e r i s t i c s o f T r a n s p l a n t e d T i s s u e T i s s u e s vary g r e a t l y i n t h e i r a b i l i t y  transplantation.  The degree o f c e l l u l a r i t y 83  t o s u r v i v e a f t e r homo-  and v a s c u l a r i t y  of the  TABLE XVII.  A summary of major experiments concerning immune responses i n W mutant mice.  Experiment  Results  S t a t i s t i c a l Test  1. Skin a l l o g r a f t s examined h i s t o l o g i c a l l y 8 days a f t e r transplantation.  Graft rejection scores of W/W mice are higher than those of +/+ mice.  Mann-Whitney U-test p = .0036  2. MST of skin a l l o g r a f t s exchanged across strong histocompatibility barriers.  +/+: MST = 9.7 days W/WV: MST =9.3 days (no s i g n i f i c a n t difference)  L i t c h f i e l d and Wilcoxon method f o r evaluating dose-effect experiments.  3. MST of skin a l l o g r a f t s exchanged across weak h i s t o compatibility b a r r i e r s .  +/+: MST = 20-22 days W/W: MST appears to be less than that f c r +/+ mice.  L i t c h f i e l d and Wilcoxon method f o r evaluating dose-effect experiments.  4. Uptake of t r i t i a t e d thymidine by peripheral blood c e l l s of mice bearing skin allografts.  8 days a f t e r transplantation, counts i n W/W cultures are higher than counts i n +/+ cultures.  Student's t - t e s t p = < .05  5. Agglutinin production in response to immunization with SRBC  1° response: no difference between +/+ and W/W mice. 2° response: t i t e r s i n W/W mice are higher than t i t e r s i n +/+ mice.  6. PFC production i n response to immunization with SRBC.  W/W mice produce fewer PFC than do +/+ mice. W/+ and W/+ mice produce PFC i n t e r mediate i n number between those of +/+ and those of W/W mice.  v  v  v  v  v  v  Student's t - t e s t p = < .05 Mann-Whitney U-test p - .014 Kruskall-Wallis One-Way Analysis of Variance p = < .02 continued  TABLE XVII.  (Continued)  Statistical  Experiment  Results  7. Uptake o f t r i t i a t e d t h y midine by s p l e e n c e l l s s t i m u l a t e d w i t h LPS or Con-A.  LPS: W/W c u l t u r e s take up fewer counts and e x h i b i t lower s t i m u l a t i o n i n d i c e s than do +/+ c u l t u r e s . Con-A: W/W c u l t u r e s take up more counts a t 96 hours than do +/+ c u l t u r e s but do n o t e x h i b i t h i g h e r stimulation indices. V  Test  Mann Whitney U - t e s t p = .05  v  cn Ul  86  tissue, i t s mitotic activity  upon h e a l i n g - i n ,  the n a t u r e and  abundance  of i t s ground s u b s t a n c e , and  the q u a n t i t a t i v e  e x p r e s s i o n of  transplan-  t a t i o n a n t i g e n s on graft. as  i t s c e l l surfaces  a l l a f f e c t s u r v i v a l of  A l l o g r a f t s of bone, e n d o c r i n e g l a n d s , and  liver,  f o r example, may  donor and  histocompatibility donor and  show p r o l o n g e d or i n d e f i n i t e s u r v i v a l i f  one  general"  d i f f e r e n c e s , and w i l l not  and  S i l v e r s , 19 71,  survive  Skin  unduly p e s s i m i s t i c p i c t u r e of  (Billingham  loci.  Skin,  of the most s e n s i t i v e o f a l l t i s s u e s  h o s t are matched a t a l l H l o c i .  to p r o v i d e "an  the  c e r t a i n organs such  h o s t a r e matched at major h i s t o c o m p a t i b i l i t y  however, appears to be  in  may  the  to  i n d e f i n i t e l y unless  g r a f t s , i n f a c t , seem f a t e of a l l o g r a f t s  p. 91).  This  sensitivity,  however, makes s k i n g r a f t s i d e a l l y s u i t e d f o r i n v e s t i g a t i o n s where s u b t l e e f f e c t s on  the  a l l o g r a f t response are  2) H i s t o c o m p a t i b i l i t y Tissue  constitute can  in  the  i s governed by  a number o f genes which  components ( S n e l l e t a l . , 1973).  transplantation  transplantation  These  a n t i g e n s which, i f f o r e i g n to the  i n c i t e immune r e s p o n s e s .  i f a l l the  detected.  Genes  compatibility  code f o r c e l l s u r f a c e  to be  structures host,  Permanent g r a f t acceptance o c c u r s  a n t i g e n s p r e s e n t i n the  g r a f t are  also present  host. The  t h a t has  H-2  locus  of  the mouse i s a major h i s t o c o m p a t i b i l i t y  r e c e n t l y been r e c o g n i z e d as  c o n s i s t i n g o f a number of  l i n k e d genes which c o n t r o l a v a r i e t y of immunological phenomena ler H-2K  only  and and  D a v i d , 19 75). H-2D,  At  which p l a y  e i t h e r end  of the H-2  region  are  two  the predominant r o l e i n d e t e r m i n i n g  complex  tightlyr (Shreffloci,  87  transplantation serologically  immunity i n mice.  defined  by  d i v e r s i t y of a n t i b o d i e s ; polypeptide portions  The  their ability the K and  of two  a l l e l e s a t these l o c i to engender and  be  react with  D a l l e l e s appear to code f o r  glycoprotein  from c e l l membranes ( S h r e f f l e r and  can  m o i e t i e s which can be  D a v i d , 1975).  Strong  the isolated  rejection  responses are e l i c i t e d i n s k i n g r a f t s exchanged between animals d i f f e r e n t K and p r o d u c t s of antigens.  D alleles,  i n d i c a t i n g that  these genes are p r o b a b l y the The  b i o l o g i c a l function  however, i s unknown ( S h r e f f l e r and  the  (Bailey, Grafts  1963;  f a c t o r s , play B a i l e y and  s e r o l o g i c a l l y detectable  of these genes and  t h e i r products,  D a v i d , 1975). and  Y-linked  a lesser role i n transplantation  Hoste, 1970;  Stimpfling  and  f o r prolonged p e r i o d s b e f o r e f i n a l l y b e i n g r e j e c t e d . genes appear to a c t c u m u l a t i v e l y , so  i s q u i c k e r when donor and  histoimmunity.  Reichert,  exchanged between animals d i f f e r i n g a t minor l o c i may  compatibility  carrying  classic histocompatibility  A number of minor H genes, i n c l u d i n g Xcompatibility  a  1971).  survive  These weaker h i s t o that  h o s t d i f f e r a t more than one  graft minor  rejection locus.  3) Other Genes A f f e c t i n g Immune Responses The depends not H-2D  of the  immune response e l i c i t e d by  only upon d i s p a r i t y between donor and  l o c i , but  complex and so  severity  a l s o upon the  elsewhere i n the  f a r described  control  to s p e c i f i c a n t i g e n s  the  an a l l o g r a f t  h o s t a t H-2K  and  activity  of o t h e r genes b o t h i n the  H-2  genome.  Most l r (immune response) genes  amount of a n t i b o d y produced i n response  (Gunther, 1973;  Mozes and  S h e a r e r , 1972),  but  88  genes c o n t r o l l i n g s u s c e p t i b i l i t y  to t h y r o g l o b u l i n autoimmunity, t o l e r a n c e  i n d u c t i o n to BSA,  and the p r o d u c t i o n o f r e a g i n s have a l s o been  (Gunther, 1973).  I n a d d i t i o n , two l o c i  have been r e p o r t e d :  a dominant, H-2  defined  r e g u l a t i n g a l l o g r a f t reaponsos  l i n k e d gene has been shown to  govern the response of female mice to the male H-Y  antigen  (Bailey  and H o s t e , 1970; G a s s e r and S i l v e r s , 1971), and a dominant, non-U-2 l i n k e d gene appears t o govern the r e j e c t i o n o f a l l o g e n e i c bone marrow cells  i n mice (Cudkowicz, 19 71).  antigens are l i k e l y I r genes may They may,  Immune r e s p o n s e s to most i f not a l l  t o be r e g u l a t e d by one o r more I r genes. a c t i n many d i f f e r e n t ways t o control immune response  f o r example, a f f e c t a n t i g e n p r o c e s s i n g , a n t i g e n  the number of s p e c i f i c a l l y  reacting c e l l s ,  recognition,  a c t i v a t i o n and p r o l i f e r a t i o n ,  immunoglobulin p r o d u c t i o n , and so on (Gunther, 19 73).  McDevitt  (19 75)  has r e p o r t e d t h a t a t l e a s t some I r genes a r e a l s o r e s p o n s i b l e f o r controlling T-cell-B-cell interaction. t h e i r mechanisms  The f u n c t i o n s of I r genes and  o f a c t i o n , however, a r e a p p a r e n t l y complex and a r e  o n l y b e g i n n i n g t o be i n v e s t i g a t e d ; a b e t t e r u n d e r s t a n d i n g o f I r gene a c t i v i t y seems fundamental to a b e t t e r u n d e r s t a n d i n g o f immune p r o c e s s e s in  B.  general.  R e c o g n i t i o n and S e n s i t i z a t i o n  1) A n t i g e n R e c o g n i t i o n by  Lymphocytes  The i n i t i a l event i n the development o f t r a n s p l a n t a t i o n is  antigen recognition,  the event by which immunocompetent h o s t  Immunity cello  89 first  i n t e r a c t with foreign material  ( F i g u r e 16).  This process i s  a f u n c t i o n of a n t i g e n - s e n s i t i v e s m a l l lymphocytes, and mediated by  immunoglobulin r e c e p t o r s  et a l . , 1973;  Greaves, 1970;  on  D i e n e r and  the  lymphocyte s u r f a c e  Langman, 1975).  o f the i n t e r a c t i o n between a n t i g e n - s e n s i t i v e c e l l s of the g r a f t , however, and reactive cells  are  i s probably  and  The the  the p r e c i s e mechanism by which  t r i g g e r e d , are not  c l e a r (Diener  and  (Altman  nature antigens  antigen-  Langman, 19 75).  2) S e n s i t i z a t i o n The has  response of the  lymphoid system to a n t i g e n i c  stimulation  been w e l l documented (Andre e t a l . , 1962a, 1962b; Andre-Schwartz,  1964;  S c o t h o r n e and  Within  Scothorne,  t w e n t y - f o u r hours a f t e r a l l o g r a f t  detectable in cell  McGregor, 1955;  i n the  t r a n s p l a n t a t i o n , changes  d r a i n i n g lymph node, and by  d i v i s i o n i s apparent  ( D e v l i n and  1957; P i e r c e , 1967).  72 hours a marked  Ramm, 19 71).  By  increase  4 days, c l u s t e r s  o f l a r g e p y r o n i n o p h i l i c c e l l s are p r e s e n t  i n the node ( P a r r o t t and  Sousa, 1966)  are a p p e a r i n g i n the  (Hall,  and  1967).  By  a c t i v a t e d lymphoid c e l l s the s i x t h day  after transplantation, cells  of t r a n s f e r r i n g immunity are p r e s e n t p e r i p h e r a l blood lymphoid c e l l s (Figure  respond to a n t i g e n ,  lymph  the  i n which  i s the p r o c e s s of s e n s i t i z a t i o n  16). With r e g a r d  to s k i n a l l o g r a f t s , a number of agents, i n c l u d i n g  "passenger" l e u k o c y t e s ,  epidermal c e l l s ,  endothelium, have been suggested as gens .  This process,  de  capable  i n s i g n i f i c a n t numbers i n  ( B i l l i n g h a m e_t a l . , 1962).  are  f i b r o b l a s t s , and  the  vascular  the s o u r c e o f the s e n s i t i z i n g a n t i -  90 ure  16.  A s i m p l i f i e d and s c h e m a t i c r e p r e s e n t a t i o n of some o f the events o c c u r r i n g d u r i n g the course o f a c e l l u l a r immune response. These a r e d i s c u s s e d i n d e t a i l i n the t e x t .  Recognition: Antigen-sensitive c e l l s interact with graft How and where t h i s occurs i s n o t known. S m a l l lymphocytes may e n t e r the g r a f t , i n t e r a c t w i t h g r a f t and r e t u r n t o lymph nodes.  antigens.  antigens,  Macrophages may i n t e r a c t w i t h g r a f t a n t i g e n s and r e t u r n to lymph nodes, perhaps c a r r y i n g g r a f t a n t i g e n s bound t o t h e i r s u r f a c e s . G r a f t a n t i g e n s may e n t e r l y m p h a t i c v e s s e l s nodes.  and t r a v e l t o lymph  Sensitization: Lymphoid c e l l s which have been s p e c i f i c a l l y a c t i v a t e d by g r a f t a n t i g e n s " t r a n s f o r m " i n t o l a r g e , h y p e r b a s o p h i l i c cells. C e l l d i v i s i o n occurs. A c t i v a t e d c e l l s are released i n t o the lymph and e v e n t u a l l y e n t e r the b l o o d stream.  Graft Rejection: S p e c i f i c a l l y a c t i v a t e d lymphocytes e n t e r the g r a f t and may, upon i n t e r a c t i o n w i t h g r a f t a n t i g e n s , r e l e a s e v a r i o u s lymphokines: MIF and ECF cause macrophages and e o s i n o p h i l s t o accumulate i n the a r e a ; TF and LAF a c t i v a t e o t h e r lymphocytes which may then a l s o r e l e a s e lymphokines; LT may e x e r t a c y t o t o x i c e f f e c t on g r a f t cells. ECF LAF LT MIF TF  -  e o s i n o p h i l chemotactic f a c t o r lymphocyte a c t i v a t i o n f a c t o r lymphotoxin macrophage i n h i b i t i o n f a c t o r transfer factor  91  Spec if ical ly Sensitized Lymphocytes Inter act w i t h G r a f t Antigens  LT MIF ECF TF.LAF (  Host Lymphatic Vesse' Effector A r m of Immune Response  Afferent a r m of C e l l u l a r I mmune Respon Transformation of Smal Lymphocytes intd Specifically Sensitized Lymphocytes  Sensitized i Lymphocytes J Enter Blood -j Stream C e n t r a l A r m of Immune Response •VV ,  B  l  o  o  d  92  The o r i g i n of the vasculature i n skin grafts and whether or not i t is, antigenic has long been a subject of controversy.  Although  i n orthotopic f u l l - t h i c k n e s s skin grafts i n rabbits and i n heterotopic grafts of hamster cheek pouch s k i n , the o r i g i n a l g r a f t vessels appear to constitute the permanent vasculature of the graft (Lambert,  1971;  H a l l e r and Billingham, 1964), other reports i n d i c a t e that i n rats and mice the graft vessels do not become functional again; instead,  new  vessels from the host appear to grow i n along the pre-existing g r a f t vessels (Converse and Ballantyne, 1962; et a l . , 1967;  Zarem, 1969).  Converse e_t a l . , 1965;  Zarem  I f blood vessels i n these grafts are indeed  of host o r i g i n , the vascular endothelium can not be the source of a n t i genic stimulation. The small population  of "passenger" leukocytes normally c a r r i e d  by an allogeneic skin g r a f t , on the other hand, does appear to be t i r e l y s u f f i c i e n t to s e n s i t i z e the host and Hart, 1971;  (Steinmuller, 1969;  Barker and Billingham, 1972).  en-  Steinmuller  A l l o g r a f t s of pure e p i -  dermis, however, which are l a r g e l y devoid of passenger leukocytes,  are  rejected almost as quickly as f u l l - t h i c k n e s s skin grafts (Billingham and Sparrow, 1954).  Epidermal c e l l s , therefore, probably play  the  major role i n i n i t i a t i n g skin graft s e n s i t i v i t y , and passenger c e l l s at most a minor role (Billingham, 19 71).  In addition, the f i b r o b l a s t  population undoubtedly c a r r i e s transplantation antigens, and may  also  contribute to g r a f t a n t i g e n i c i t y . Just where the i n i t i a l confrontation between lymphocyte and antigen takes place i s another point of uncertainty.  Sensitization  93  may o c c u r p e r i p h e r a l l y b l o o d move through posed  as i m m u n o l o g i c a l l y  the v e s s e l s  to g r a f t antigens.  t r a l l y , with antigenic borne c e l l s  from the g r a f t  Intact  from t h e  o r parenchyma o f the g r a f t and a r e ex-  Alternatively,  material  a c t i n g w i t h lymphocytes  competent c e l l s  s e n s i t i z a t i o n may o c c u r  draining  cen-  o r b e i n g c a r r i e d by lymph-  to the l o c a l lymph node and t h e r e i n t e r -  (Medawar, 1958).  l y m p h a t i c d r a i n a g e has been proven n e c e s s a r y f o r r a p i d  s e n s i t i z a t i o n t o take p l a c e (Lambert ham, 1967, 1968).  Thus, f i r s t - s e t  e t a l . , 1965; B a r k e r and B i l l i n g -  s k i n g r a f t s which have a normal  b l o o d s u p p l y b u t no l y m p h a t i c d r a i n a g e a r e n o t r e a d i l y f a i l t o s e n s i t i z e the h o s t .  Lymph d r a i n i n g  r e j e c t e d and  the s i t e o f s k i n  allografts  c o n t a i n s much c e l l d e b r i s ( H a l l , 1967), and c h e m i c a l s such as f l u o r o d i n i t r o b e n z e n e appear to combine w i t h s o l u b l e of a p p l i c a t i o n ,  19 71).  Thus, a n t i g e n i c  material  the l y m p h a t i c s and s e n s i t i z e c e l l s w i t h i n Peripheral alymphatic g r a f t s  exhibit  act with g r a f t antigens  (Tilney  and F o r d , 1974). that  down  the lymph nodes. Although  move through  sensi-  t h e g r a f t and i n t e r -  and Gowans, 19 71; F u t r e l l and Myers, I n each o f these s t u d i e s ,  however, the  g r a f t a n t i g e n s may have reached lymphoid  the b l o o d s t r e a m can n o t be e x c l u d e d . tization actually  t o pass  p r o l o n g e d s u r v i v a l , they do e v e n t u a l l y  as lymphocytes  possibility  appears  s e n s i t i z a t i o n , however, may i n d e e d o c c u r .  t i z e the h o s t , p o s s i b l y  Tilney  a t the s i t e  then e n t e r t h e lymph and flow i n t o l o c a l lymph nodes  ( H a l l and Smith,  1972;  proteins  can o c c u r , t h e r e f o r e ,  tissue v i a  Whether o r n o t p e r i p h e r a l remains  i n doubt.  sensi-  C.  The R e j e c t i o n P r o c e s s  1) Role of  Lymphocytes  Lymphocytes  have l o n g been s u s p e c t e d o f f u n c t i o n i n g i n the  d e s t r u c t i o n o f tumours and s o l i d  tissue allografts.  Almost  invariably,  r e j e c t i o n of f o r e i g n t i s s u e i s a s s o c i a t e d w i t h the movement o f mononuclear c e l l s  i n t o the g r a f t .  That i t i s the s m a l l lymphocyte, however,  which i s the key c e l l concerned w i t h g r a f t  r e j e c t i o n has been demon-  s t r a t e d i n a v a r i e t y o f ways. Allograft  r e j e c t i o n e i t h e r does not o c c u r o r i s v e r y p r o l o n g e d  i n a n i m a l s d e p l e t e d o f s m a l l lymphocytes. for  T h i s has been shown i n r a t s  donor-host combinations d i f f e r i n g a t weak h i s t o c o m p a t i b i l i t y  (McGregor  and Gowans, 1964;  Roser and F o r d , 1972), and i n mice  loci  (Deaton  and Bach, 1970), a f t e r c h r o n i c d r a i n a g e o f lymphocytes from the t h o r a c i c duct, o r a f t e r treatment w i t h l y m p h o c y t o t o x i c a g e n t s .  X-irradiation  and treatment w i t h drugs which s u p p r e s s the p r o d u c t i o n o f lymphocytes a l s o p r o l o n g a l l o g r a f t s u r v i v a l t o a degree dependent of  the treatment (see, f o r example,  (nude) mice 1961)  ( K i n d r e d , 1971)  B r e n t , 1958).  on t h e s e v e r i t y  Congenitally  and n e o n a t a l l y thymectomised  mice  athymic  (Miller,  a r e a l s o s e v e r e l y d e f i c i e n t i n lymphocytes, and e x h i b i t d e p r e s s e d  immune r e s p o n s e s . Cell cyte i n g r a f t cells  t r a n s f e r experiments have a l s o i m p l i c a t e d rejection.  causes f i r s t - s e t  the s m a l l lympho-  A d o p t i v e t r a n s f e r o f s e n s i t i z e d lymph node  skin allografts  to be r e j e c t e d i n s e c o n d - s e t  time ( B i l l i n g h a m e_t a l . , 1954a), and t r a n s f e r o f normal, lymph node c e l l s  unsensitized  can b r i n g about the r e j e c t i o n of l o n g - s t a n d i n g  grafts  95  on  t o l e r a n t animals  duct c e l l s  and  ( B i l l i n g h a m ex al.,  p e r i p h e r a l blood  c e l l s are as  immunity as are lymph node c e l l s 1962). and  1956).  c a p a b l e of t r a n s f e r r i n g  (Gowans e_t aJL. , 1961;  F i n a l l y , s t u d i e s on g r a f t - v e r s u s - h o s t  B r e n t , 1959;  Gowans, 1962;  syndrome.  l i s h such phenomena as a l l o g r a f t  2) The  Cellular  small  et a l . ,  (Billingham.  have shown t h a t  competent c e l l s  E x a c t l y how  Billingham  reactions  Hildemann, 1964)  lymphocytes are the i m m u n o l o g i c a l l y the g r a f t - v e r s u s - h o s t  In a d d i t i o n , t h o r a c i c  small  responsible lymphocytes  r e j e c t i o n , however, i s not  for accomp-  clear.  Infiltrate  A d i a g n o s t i c f e a t u r e of a l l o g r a f t s undergoing r e j e c t i o n i s accumulation w i t h i n and  the g r a f t of l a r g e numbers o f w h i t e b l o o d  most p a r t i c u l a r l y  of mononuclear c e l l s .  I t has  been  the  cells,  generally  assumed t h a t t h i s c e l l u l a r i n f i l t r a t e i s the means by which g r a f t r e j e c t i o n i s accomplished.  As  p a r t o f an e x t e n s i v e  study of  r e s p o n s e s , J . B. Murphy (1926) i n v e s t i g a t e d the e f f e c t of or s t i m u l a t i o n of the lymphoid system on c o n c l u d e d t h a t the the  infiltrating  [ r e j e c t i o n ] mechanism e x e r t s  c e l l s "are  t e r i z e and  identify  agreement as and  Shorter  leukocytes, allografts reticulum  to the  these c e l l s .  and  of a l l o g r a f t  rejection, uticharac-  however, been i n c o m p l e t e  i d e n t i t y of the c e l l s i n the i n f i l t r a t e . that n e u t r o p h i l s , other  h i s t i o c y t e s are  and  the agent through which  There has,  Titus  polymorphonuclear  the predominant c e l l  undergoing r e j e c t i o n , but cells  r e j e c t i o n process,  e l e c t r o n m i c r o s c o p y , have attempted t o  (1962) r e p o r t and  depletion  i t s force."  A number of h i s t o l o g i c a l s t u d i e s l i z i n g b o t h l i g h t and  the  allograft  type i n s k i n  A l l e g r a e_t al_. (1968) r e p o r t  lymphocytes predominate.  that  Weiner ejt a l . (1964) r e p o r t  96  t h a t mononuclear  c e l l s b e a r i n g some u l t r a s t r u c t u r a l s i m i l a r i t i e s  lymphocytes and monocytes a r e the predominant  to both  c e l l associated with  allo-  g r a f t r e j e c t i o n , and, f o r want o f a more a c c u r a t e name, they term t h e s e cells  graft  rejection cells.  T i t u s and S h o r t e r , however, were w o r k i n g  w i t h mice, A l l e g r a ejt a l . w i t h monkeys, and Weiner s p e c i e s d i f f e r e n c e s may account i n p a r t the o b s e r v a t i o n s o f these workers;  et. al_. w i t h r a b b i t s ;  f o r the l a c k o f agreement  I n a d d i t i o n , the i d e n t i f i c a t i o n  of many hematogenous c e l l s by m o r p h o l o g i c a l c r i t e r i a i s a t b e s t and i s c o m p l i c a t e d by a l a c k o f s t a n d a r d nomenclature. v e r s develop d i f f e r e n t  different  infiltrate  (19 74) u s i n g the t e c h n i q u e s o f J a k o b i s i a k  e t a l . (1971) t o i s o l a t e e n z y m a t i c a l l y skin allografts.  from  comparable.  A unique study o f the c o m p o s i t i o n o f the c e l l u l a r has been performed by C o l l e n  difficult,  D i f f e r e n t obser-  c l a s s i f i c a t i o n schemes and r e s u l t s  laboratores are not r e a d i l y  among  the c e l l s  i n f i l t r a t i n g mouse  C o l l e n f i n d s t h a t lymphocytes, n e u t r o p h i l s , and macro-  phages make up the g r e a t e r p r o p o r t i o n o f the i n f i l t r a t e ; among these c e l l s , she has i d e n t i f i e d t h r e e c l a s s e s o f lymphocytes:  typical  small  lymphocytes, t y p i c a l l a r g e lymphocytes, and t r a n s f o r m e d lymphocytes. C o l l e n a l s o i d e n t i f i e d a group o f b a s o p h i l i c c e l l s which she b e l i e v e d were p r o b a b l y a l s o lymphoid i n n a t u r e b u t were d i s t i n g u i s h e d from o t h e r s m a l l and l a r g e lymphocytes by t h e i r h y p e r - b a s o p h i l i c c y t o p l a s m . these v a r i o u s c e l l s  come t o be l o c a l i z e d i n the g r a f t  and what  How  their  s p e c i f i c f u n c t i o n s a r e , however, i s u n c e r t a i n . I t has been g e n e r a l l y assumed t h a t among the c e l l s which a c c u mulate i n a l l o g r a f t s  are s p e c i f i c a l l y  a c t i v a t e d lymphocytes  t h a t were  produced  i n l o c a l lymph nodes and r e l e a s e d i n t o  the blood.  These  cells  are presumed to "home" i n t o the a l l o g r a f t and d e s t r o y i t ( F i g u r e 1 6 ) . Prendergast  (1964) l a b e l l e d i n v i v o those c e l l s o r i g i n a t i n g  from a  lymph node d r a i n i n g t h e s i t e o f an a l l o g r a f t , and found t h a t many o f them d i d l o c a l i z e i n the g r a f t bed.  The l o c a l i z a t i o n , however, d i d  n o t appear t o be s p e c i f i c ; l a b e l l e d c e l l s e n t e r e d g r a f t s from u n r e l a t e d donors w i t h e q u a l f a c i l i t y . cells  Hall  (1967) l a b e l l e d i n v i t r o  lymphoid  c o l l e c t e d from the e f f e r e n t lymph o f a lymph node d r a i n i n g the  s i t e o f an a l l o g r a f t , of t h e h o s t a n i m a l .  then r e i n j e c t e d  the c e l l s i n t o  the c i r c u l a t i o n  He found t h a t two t o t h r e e l a b e l l e d c e l l s out  o f e v e r y 1,000 i n j e c t e d e n t e r e d the homograft, whereas almost none e n t e r e d an a u t o g r a f t on the same a n i m a l . g i n a t e d i n response  cells  t h a t had o r i -  t o a b a c t e r i a l a n t i g e n , however, e n t e r e d the homo-  graft with equal f a c i l i t y .  Lance and Cooper (1972), on t h e o t h e r hand,  u s i n g a unique double l a b e l l i n g of a c t i v a t e d c e l l s  Labelled  t e c h n i q u e , found t h a t s m a l l numbers  d i d appear t o home s p e c i f i c a l l y  had s t i m u l a t e d t h e i r p r o d u c t i o n . localization i s specific;  i n t o the g r a f t w h i c h  These workers a r e c o n v i n c e d  that  the n e g a t i v e r e s u l t s o f H a l l and P r e n d e r g a s t  they f e e l a r e due t o t h e d i f f i c u l t y o f l a b e l l i n g o n l y t h e s p e c i f i c a l l y a c t i v a t e d c e l l s , which a r e p r o b a b l y v e r y few i n number. even i n Lance and Cooper's experiments,  Labelled  formed o n l y a s m a l l  of the c e l l u l a r i n f i l t r a t e a s s o c i a t e d w i t h g r a f t r e j e c t i o n . and F o r d  (19 74) a l s o found "some degree  s e n s i t i z e d lymphocytes  cells,  percentage Tilney  o f s e l e c t i v e accumulation o f  i n specific allografts  o f s k i n " b u t warned  "the d i f f e r e n c e s were s m a l l and s h o u l d be c a u t i o u s l y  interpreted."  that  98  3) E f f e c t o r Mechanisms i n A l l o g r a f t a)  Specificity One  Rejection  of g r a f t r e j e c t i o n  o f the most s t r i k i n g c h a r a c t e r i s t i c s of s k i n  r e j e c t i o n i s i t s remarkable s p e c i f i c i t y . M i n t z and  S i l v e r s (19 70)  utilizing  the s e l e c t i v e d e s t r u c t i o n confines was  b l a s t s and  experiment  a l l o p h e n i c mice has  s t r a i n mice, only  the h o s t s u r v i v e d . of c e l l s  d e s t r o y e d as  of the  p o r t i o n was Any  S i l v e r s d i d note t h a t  specificity  i f the  as  majority  able  complete w i t h i n  two  weeks and  to account f o r such  the  no  further necrosis  destruction  they o f f e r to the  i n vivo process.  t o x i c i t y have demonstrated t h a t Those most l i k e l y  cell-mediated-cytotoxicity  and  larger foreign  occurred.  of s k i n a l l o g r a f t s  specificity.  s t a g e s of a l l o g r a f t r e j e c t i o n i n v i v o , and  mechanisms.  I f the  g r a f t were a c c e p t e d , however, then r e j e c t i o n o f the  A number o f i n v i t r o models are b e l i e v e d  clues  specificities  a r e s u l t of n o n s p e c i f i c n e c r o s i s .  mechanism p r o p o s e d t o e x p l a i n  must be  donors  i n the g r a f t were f o r e i g n , then the e n t i r e g r a f t would even-  t u a l l y be part  M i n t z and  the narrow  those melano-  c e l l s which c a r r i e d f o r e i g n H-2  were d e s t r o y e d ; those c e l l s which were of the same H-2  by  demonstrated  When s k i n from a l l o p h e n i c  to i s o g e n i c p a r e n t a l  hair f o l l i c l e  elegant  of i n d i v i d u a l f o r e i g n c e l l s w i t h i n  of s i n g l e h a i r f o l l i c l e s .  transplanted  An  allograft  to r e p r e s e n t  have been s t u d i e d Studies  t a r g e t c e l l s may  of i n v i t r o be  k i l l e d by  to f u n c t i o n i n v i v o are  various for  the  cytoseveral  contact-induced  antibody-dependent c y t o t o x i c i t y , b o t h  of which are s p e c i f i c c y t o t o x i c mechanisms.  The  i n c y t o t o x i c mechanisms, however, must a l s o be  r o l e of  lymphokines  considered.  99  b) Contact-induced cell-mediated-cytotoxicity It has been generally assumed that s p e c i f i c a l l y activated lymphocytes accumulate i n a l l o g r a f t s and exert a d i r e c t and  specific  cytotoxic e f f e c t either on the vasculature of the graft or on the graft epithelium i t s e l f .  Lymphocytes from s e n s i t i z e d donors have been shown  to be s p e c i f i c a l l y cytotoxic for target c e l l s carrying the s e n s i t i z i n g antigens  (Govaerts,  1960;  mann and Holm, 1969).  Rosenau and Moon, 1961; Wilson, 1963;  Such c e l l s cultured i n v i t r o with target  monolayers are reported to c l u s t e r around the target c e l l s , them to cease DNA  Perlcell  causing  synthesis and to eventually round up, become detached,  and die (Wilson, 1967;  Ax et a l . , 1968).  Berke (1972) reports that  s e n s i t i z e d c e l l s which are able to destroy tumour c e l l s i n v i t r o are also able to retard tumour growth i n vivo, and Wagner et a l . (19 72) report that c e l l s s e n s i t i z e d in. v i t r o are able to b r i n g about the r e j e c tion of tumour and skin a l l o g r a f t s i n vivo. mediated-cytotoxicity,  Contact-induced c e l l -  therefore, as demonstrated i n v i t r o , i s thought  to play a major role i n the r e j e c t i o n of a l l o g r a f t s i n vivo. The work of Berke (1972) and Wagner et a l . (1972) demonstrates that the c e l l s responsible for i n v i t r o c y t o t o x i c i t y are also functional i n vivo; the precise mechanism by which they induce graft destruction, however, i s not necessarily the same i n both systems.  Indeed, the  death of skin grafts i s not, i n f a c t , r e a d i l y ascribed to contactinduced c y t o t o x i c i t y directed against the vascular endothelium or against the e p i t h e l i a l c e l l s themselves.  Vascular s t a s i s has been  reported to occur early i n the r e j e c t i o n process and has often been  100  c i t e d as the main cause of graft necrosis  (Rolle, 1959; Waksman, 19 74).  The vasculature of mouse skin g r a f t s , however, i s probably of  host  o r i g i n (p. 92) and therefore can not be the primary target of s p e c i f i c e f f e c t o r mechanisms.  In addition, skin i s remarkably r e s i s t a n t to  ischemia and can survive  f o r long periods i n v i t r o or i n the anterior  chamber of the eye where no vascular supply exists  (Medawar, 1948).  Brent (1958) has noted that the time i n t e r v a l between vascular breakdown and e p i t h e l i a l destruction of graft  i s too b r i e f f o r ischemia to be the cause  deterioration. Neither can e p i t h e l i a l necrosis be explained by the d i r e c t  cytotoxic action of s p e c i f i c a l l y activated lymphoid c e l l s i n contact with the c e l l s of the g r a f t .  E p i t h e l i a l necrosis  i s often seen w e l l  before the invading c e l l s reach the upper layers of the g r a f t , and before any contact i s made between lymphoid c e l l s and epithelium 9).  In addition, only a few of the lymphocytes entering  appear to be s p e c i f i c f o r the graft (p. 9 7).  the graft  The concept of d i r e c t ,  contact-induced c y t o t o x i c i t y by s p e c i f i c a l l y activated therefore,  (Figure  lymphocytes,  although favoured by many as the primary mechanism respon-  s i b l e f o r a l l o g r a f t r e j e c t i o n , appears to be an o v e r s i m p l i f i c a t i o n of the r e j e c t i o n process, and even, i n the words of Billingham and S i l v e r s (1971), "a complete misrepresentation" of the actual events.  c) Role of lymphokines Sensitized gen  lymphocytes upon i n t e r a c t i o n with s p e c i f i c a n t i -  are known to release  into the medium a number of b i o l o g i c a l l y active  factors c a l l e d lymphokines (Granger and Williams, 1968).  Some of the  101  lymphokines have been f u n c t i o n a l l y inhibit  c h a r a c t e r i z e d by t h e i r a b i l i t y  the m i g r a t i o n o f macrophages (MIF, macrophage i n h i b i t i o n  to e x e r t a c h e m o t a c t i c i n f l u e n c e on macrophages and e o s i n o p h i l s and ECF, macrophage and e o s i n o p h i l c h e m o t a c t i c f a c t o r s ) , kill  cells  in vitro  formation factor) lymphocytes is  t o t r a n s f o r m and d i v i d e  (Dumonde e t a l . ,  1969;  Granger, 19 72).  sensitivity,  The a c t i v i t y  t o normal lymphocytes  o f MIF, MCF,  of the c e l l u l a r i n f i l t r a t i o n  (Lawrence,  trans-  Sensitized (TF) which  c a p a b l e of t r a n s f e r r i n g s p e c i f i c a n t i g e n i c s e n s i t i v i t y ,  homograft  (MCF  normal  (LTF, lymphocyte  a r e a l s o a b l e to produce a " t r a n s f e r " f a c t o r  factor),  to a c t u a l l y  (LT, a n o n s p e c i f i c l y m p h o t o x i n ) , and to s t i m u l a t e  lymphocytes  to  including  1973).  and ECF can r e a d i l y a c c o u n t f o r much  associated with graft  rejection.  s i t i z e d h o s t lymphocytes which accompany t h i s i n f i l t r a t e may  Unsenbe  con-  v e r t e d t o a s p e c i f i c a n t i g e n - r e a c t i v e s t a t e by TF, such t h a t they then behave as s p e c i f i c a l l y  sensitized cells:  they t r a n s f o r m and r e l e a s e lymphokines. sitized  upon i n t e r a c t i o n w i t h a n t i g e n , A v e r y few s p e c i f i c a l l y  sen-  lymphocytes, then, e n t e r i n g the g r a f t , i n t e r a c t i n g w i t h g r a f t  a n t i g e n s , and r e l e a s i n g TF, c o u l d c o n v e r t many uncommitted  h o s t lympho-  c y t e s t o a n t i g e n r e a c t i v e s t a t e s such t h a t they too would be a b l e to function i n graft  destruction.  Upon i n t e r a c t i o n w i t h a n t i g e n , b o t h s e n s i t i z e d and lymphocytes  r e c r u i t e d by  ( L T ) , a lymphokine for target c e l l s  the a c t i o n o f TF e l a b o r a t e  lymphocytes lymphotoxin  t h a t has been shown to be n o n - s p e c i f i c a l l y  in vitro  (Granger and W i l l i a m s , 1968,  and Lucas, 1973; Kramer and Granger, 1975).  1971;  Walker and Lucas  cytotoxic Walker (1973)  102  and  Kramer and  Granger (19 75)  have s u g g e s t e d t h a t s p e c i f i c i n v i t r o  c e l l - m e d i a t e d - c y t o t o x i c i t y i s a l s o mediated by has  LT,  and  Granger  (1969)  s u g g e s t e d t h a t LT i s the p r i n c i p a l means by which s p e c i f i c g r a f t  d e s t r u c t i o n i s accomplished i n v i v o  ( F i g u r e 16).  The  p r e c i s e mechanism  of the c y t o t o x i c r e a c t i o n , however, i s f a r from c l e a r . (19 74) have r e p o r t e d t i o n can be production  that  c e l l - m e d i a t e d - c y t o t o x i c i t y and  experimentally  dissociated:  of lymphokines r e p r e s e n t  functions.  The  Henney e_t a l . mediator produc-  t h a t c y t o t o x i c i t y and  independent c e l l - m e d i a t e d  the immune  r o l e of lymphotoxin i n c e l l - m e d i a t e d - c y t o t o x i c i t y i n  v i t r o i s , therefore, s t i l l  uncertain,  and  i t s role i n vivo subject  to  much s p e c u l a t i o n .  d) Role of The not  only  the  formation  antigens  clear.  species  and  a n t i b o d y may at a l l .  coded f o r by  h i s t o c o m p a t i b i l i t y genes  t r a n s p l a n t a t i o n immunity but of antibodies  o f the a l l o g r a f t not  antibody  i s now  humoral immunity as w e l l .  generally  s t r a i n s of animals i n v o l v e d , e i t h e r prolong  A number of e a r l y experiments  s k i n g r a f t s , but Winn (19 70) have not  quantity and  o f the s e r a ,  the k i n d s  the  of g r a f t s used,  (reviewed by  Stetson,  1963)  can b r i n g about r e j e c t i o n of  i s s k e p t i c a l of these experiments because  been h i g h l y r e p r o d u c i b l e .  b o d i e s are s u c c e s s f u l i n c a u s i n g a c u t e and  r e g a r d e d as a normal p a r t  or c u r t a i l g r a f t s u r v i v a l , or have no e f f e c t  seem to i n d i c a t e t h a t humoral a n t i b o d i e s  to be  Although  response, the r o l e of a n t i b o d y i n g r a f t r e j e c t i o n i s  Depending upon the source and  the o b s e r v a t i o n s  incite  Even when a n t i -  r e j e c t i o n , the p r o c e s s seems r a r e l y  the g r a f t s p e r s i s t f o r s e v e r a l weeks b e f o r e  being  103  sloughed.  Jooste et_ a l . (19 73) have recently reported that, i n r a t s ,  skin grafts are susceptible to damage by humoral antibodies only during a short period about two weeks a f t e r transplantation.  Healing-in grafts  and long-standing g r a f t s , they report, are not susceptible to damage by antibodies. Although humoral antibodies are not e s s e n t i a l for a l l o g r a f t r e j e c t i o n , antibody-dependent cell-mediated l y s i s may in the rejection process.  s t i l l play a role  Target c e l l s incubated i n v i t r o with very  low t i t e r s of antibody are lysed by lymphoid c e l l s from normal donors (Perlmann and Holm, 1969).  This l y s i s requires contact between lym-  phoid c e l l and the target c e l l , and occurs with antibody  dilutions  too high to give r i s e to conventional complement-mediated l y s i s .  Low  t i t e r s of antibodies reactive against major h i s t o c o m p a t i b i l i t y antigens are undoubtedly formed i n mice bearing skin a l l o g r a f t s Davies, 1973;  (Brazenor  and  Staines et a l . , 1974), and serum from such mice has been  shown to induce cell-mediated c y t o t o x i c i t y i n v i t r o (Degiovanni Lejeune, 1973).  and  Whether or not antibody-dependent cell-mediated l y s i s  a c t u a l l y plays a role i n skin a l l o g r a f t r e j e c t i o n , however, i s very uncertain. e)  Conclusion Clearly, the mechanism by which tissue a l l o g r a f t s are de-  stroyed i s a complex and as yet poorly understood process; no hypothesis has been put forth which s a t i s f a c t o r i l y explains a l l the vations.  Sensitized lymphocytes, lymphokines, and s p e c i f i c  obser-  antibody  are able to induce c y t o t o x i c i t y i n v i t r o , but the role of these various  104 effectors i n vivo i s not at a l l clear.  S p e c i f i c skin graft r e j e c t i o n  may very w e l l be a r e s u l t of the combined a c t i v i t y of each of these s p e c i f i c mechanisms, and of other non-specific tissue-damaging processes as w e l l .  v D.  Skin A l l o g r a f t Rejection i n W/W  Mice  1) MST of F i r s t - s e t H-2 Incompatible  Grafts  There are no obvious h i s t o l o g i c a l differences between skin a l l o g r a f t r e j e c t i o n i n +/+ and W/W  mice.  The healing process, the  i n f i l t r a t i o n of the graft by mononuclear and polymorphonuclear c e l l s , and the death and replacement of the graft appear to follow s i m i l a r patterns i n both groups of mice. a l l o g r a f t s on +/+ and W/W  V  The s i m i l a r i t y of the MSTs f o r skin  mice, as determined by microscopic exami-  nation, indicates that there i s no marked difference i n graft s u r v i v a l times between the two groups.  The MST reported here of 9.7 days f o r  DBA/2 skin on C57B1/6 hosts i s , however, somewhat shorter than the MST of 10.4 days reported elsewhere ( M i c r o b i o l o g i c a l Associates, Inc., 19 72) f o r this donor-host combination.  This discrepancy i s almost  c e r t a i n l y due to the fact that graft death w i l l be apparent  microsco-  p i c a l l y sooner than i t w i l l be apparent macroscopically.  2) Graft Rejection Scores on the Eighth Day a f t e r Transplantation The d i s t r i b u t i o n s of graft r e j e c t i o n scores f o r the two groups of mice on the eighth day, however, are s i g n i f i c a n t l y d i f f e r e n t .  This  s i g n i f i c a n c e i s not borderline, but quite marked (p = < .01). Such a  105  difference  i n the  d i s t r i b u t i o n of g r a f t  cates that  a difference  e x i s t between the p o i n t of g r a f t  two  i n the  v i g o u r of g r a f t  groups of mice.  s u r v i v a l as  rejection  rejection  r e j e c t i o n process i s l o s t .  breakdown b e g i n s , and  the  the  grafts  end  d u r i n g the  control  groups.  reflect  the  suffered  by  Histological  v i g o u r of  the  the  at any  graft  of the  rejection  of g r a f t s  reaction  a l r e a d y dead can  p r o c e s s which i n t h i s way r a t h e r than s i m p l y than MST that  the  end  i n f a c t be  as  time when g r a f t  w i t h i n the  indi-  significant  the  degree of  stage a f t e r t r a n s p l a n t a t i o n ;  as  such  p o i n t must be  An  as w e l l  analysis  of  a s p e c t s of  as the the  a more s e n s i t i v e  I t i s tempting to c o n c l u d e ,  difference  a difference  criteria reactions  j u s t e n t e r i n g breakdown  utilized.  i n MST  i n the  directly  U s i n g these c r i t e r i a ,  w i t h i n each g r a f t  between +/+  v i g o u r and  and  and  damage  comparisons of a l l o g r a f t  considers several  determination alone.  a l t h o u g h t h e r e i s no  t h e r e may  be  be  response and  (Corson e t a l . , 1967).  the  end  c r i t e r i a d e s c r i b e d e a r l i e r (p. 58)  mation c o n c e r n i n g the number o f g r a f t s the magnitude of  fact  r e j e c t i o n process i n experimental  have been used b e f o r e where s e n s i t i v e have been r e q u i r e d  The  reaction  r e j e c t i o n p e r i o d may  p o i n t i n comparing the  indi-  d e t e r m i n a t i o n much i n f o r -  mation c o n c e r n i n g the  vidual  does i n  In l o o k i n g only f o r the  i s done i n MST  intensity  scores probably  the  inforand  number  rejection process analysis therefore, W/W  intensity  mice  V  of  graft  v r e j e c t i o n between the s t r o n g e r and  two  more i n t e n s e .  groups, w i t h that  of W/W  mice b e i n g  the  106  3) R e j e c t i o n  o f S k i n A l l o g r a f t s A c r o s s Weak H - B a r r i e r s  A l t h o u g h no d i f f e r e n c e  i n speed o f r e j e c t i o n was apparent when  H-2 i n c o m p a t i b l e g r a f t s were t r a n s p l a n t e d  t o +/+ and W/W  hosts, a d i f -  V  f e r e n c e i n the speed o f r e j e c t i o n o f g r a f t s b e a r i n g weak H a n t i g e n s may i n f a c t e x i s t . +/+ and W/W  female h o s t s , no s u r v i v i n g  V  W/W  V  When s k i n from male donors was t r a n s p l a n t e d  to  g r a f t s were found among t h e  h o s t s a t times when g r a f t s t o +/+ h o s t s were almost a l l s t i l l  alive.  These d a t a suggest t h a t  t h e s u r v i v a l time o f s k i n  transplanted v  a c r o s s weak H b a r r i e r s may be s i g n i f i c a n t l y s h o r t e r  among W/W  hosts  than among +/+ h o s t s . 4) A Study o f t h e C e l l u l a r I n f i l t r a t e  ( C o l l e n , 1974)  A study which may f u r t h e r s u b s t a n t i a t e a c t i o n o f W/W  V  t h e more i n t e n s e r e -  mice t o s k i n a l l o g r a f t s i s t h a t o f C o l l e n  (1974).  Working  w i t h mice from t h e same s t o c k s used i n t h e experiments r e p o r t e d i n this  thesis, Collen placed  various the  s k i n a l l o g r a f t s on +/+ and W/W  V  times a f t e r t r a n s p l a n t a t i o n ,  a l l o g r a f t s by e n z y m a t i c a l l y  terized  the c e l l  graft.  Collen i d e n t i f i e d eight  she i s o l a t e d t h e c e l l s  digesting  the g r a f t s .  hosts.  At  infiltrating  She then  charac-  types and determined t h e i r r e l a t i v e numbers i n each types o f l e u k o c y t e s w i t h i n  the g r a f t s :  small,  l a r g e , and t r a n s f o r m e d lymphocytes; s m a l l  and l a r g e hyperbaso-  philic  lymphoid c e l l s ; macrophages, n e u t r o p h i l s ,  and e o s i n o p h i l s .  r e l a t i v e numbers o f these c e l l s different  V  mice was s i g n i f i c a n t l y  from t h e i r r e l a t i v e numbers i n g r a f t s on +/+ mice, on a l l  days f o l l o w i n g observation  i n g r a f t s on W/W  The  transplantation.  that  Of p a r t i c u l a r i n t e r e s t i s C o l l e n ' s  t h e number o f t r a n s f o r m e d lymphocytes i s s i g n i f i c a n t l y  107  higher i n grafts on W/W  V  hosts than i n grafts on +/+  days following transplantation.  hosts, on a l l  Transformed lymphocytes are correlated  both with the i n i t i a l response of lymphoid c e l l s to antigen and with c y t o t o x i c i t y , and are thought to represent a l l o g r a f t response. on W/W  the e f f e c t o r arm of the  Their presence i n greater numbers i n a l l o g r a f t s  hosts constitutes an i n t r i g u i n g c o r r e l a t i o n with the more severe  h i s t o l o g i c a l picture suggested by other experiments. Further consideration of these results and speculation as to /V  the cause of the apparent differences between +/+  and W/W  mice w i l l  be deferred u n t i l the end of the next section of this t h e s i s , which concerns another experiment of relevance  II.  A.  to the present discussion.  LYMPHOCYTE ACTIVATION STUDIES  General Discussion Numerous studies have described  the changes occurring i n lymph  nodes following antigenic stimulation (Scothorne, 1957; 1962;  Andre-Schwartz, 1964;  Coons, 1958;  Andre et a l . ,  Parrot and de Sousa,  1966;  Devlin and Ramm, 1971), and a few studies, mainly by H a l l (1967) and H a l l and  coworkers (1963, 1967,  c e l l population  1971)  have described  changes i n the  of the afferent and efferent lymph of lymph nodes draining  the s i t e of antigenic stimulation.  Only a few studies e x i s t , however,  of the c e l l u l a r changes i n the peripheral blood following immunization. In one of the e a r l i e s t of these, G i l l blood c e l l s from normal patients and  (1967) obtained white  from patients with drug a l l e r g i e s ,  108  and cultured these c e l l s i n v i t r o for up to f i v e days. of c e l l s undergoing spontaneous transformation was obtained  The numbers  greater i n cultures  from a l l e r g i c patients than i n cultures from healthy  subjects.  G i l l suggested that this difference was-due to increased numbers of c i r c u l a t i n g , s e n s i t i z e d lymphocytes i n the cultures from the hypers e n s i t i v e patients.  In a study of dogs bearing cardiac transplants,  Tennenbaum et a l . (1970) cultured blood lymphocytes for 24 or 72 hours. C e l l cultures from dogs bearing a l l o g r a f t s showed a 3- to 4-fold greater percentage of spontaneously transforming tures from normal dogs.  lymphocytes than did c e l l c u l -  Tennenbaum suggested that the increased  of spontaneous lymphocyte transformation  rate  i s a r e f l e c t i o n of host immuno-  competent c e l l s responding to the presence of the a l l o g r a f t . et a l . (1969) have reported that patients with  Crowther  i n f e c t i o n s or under  immunological stimulation also have increased numbers of " a t y p i c a l mononuclear c e l l s " i n the blood.  These a t y p i c a l c e l l s were i d e n t i f i e d  as plasma c e l l s , hyperbasophilic medium-sized lymphocytes, and lymphoid c e l l s , and were thought to represent  large  a population of c e l l s  derived from lymphoid tissue responding to antigenic stimulation. conclusion i s supported by H a l l and Morris  This  (1963) and H a l l (1967) who  described i d e n t i c a l c e l l s i n the lymph flowing from nodes draining s i t e s of antigenic stimulation. The numbers of a t y p i c a l mononuclear c e l l s i n the blood of immunized patients, though greater than that of normal patients, i s s t i l l low (less than 4%)  (Crowther ejt a_l. , 1969).  Their presence can be more  readily detected by measuring the uptake of l a b e l l e d DNA  precursors  109  by  these c e l l s .  Crowther et a l . (1969) r e p o r t e d  the a t y p i c a l mononuclear c e l l s l a t e d p a t i e n t s were i n DNA  i n the b l o o d  s t u d i e s of c e l l c u l t u r e s l a b e l l e d w i t h t r i t i a t e d e t a l . (1970, 1971), Dent Lee  r e p o r t a 2-  to 1 0 - f o l d i n c r e a s e  thymidine by  peripheral blood  stimu-  radioautographic thymidine.  (1970), Page et a l . (1971), and  (19 74), measuring r a d i o a c t i v i t y by  of  of i m m u n o l o g i c a l l y  s y n t h e s i s , as shown by  (  and  t h a t about 30%  scintillation  Hersh  McDonald counting,  i n the spontaneous uptake of  tritiated  lymphocytes of r e n a l t r a n s p l a n t  patients,  an i n c r e a s e which always preceded c l i n i c a l s i g n s of r e j e c t i o n by days.  Ono  e_t a l . (1972) r e p o r t s i m i l a r r e s u l t s w i t h r a t s b e a r i n g  allografts. c u r s o r s by  The cells  spontaneous uptake of r a d i o a c t i v e l y l a b e l l e d DNA from the p e r i p h e r a l b l o o d  a l l o g r a f t s appears, t h e r e f o r e , and  several  to c o r r e l a t e w i t h and Morphologically,  of humans or animals  to r e f l e c t h o s t  immunologic  heart prebearing  reactivity,  precede r e j e c t i o n c r i s e s . the  atypical cells  responsible  f o r the  uptake  o f r a d i o a c t i v e l a b e l are m o s t l y medium or l a r g e lymphoid c e l l s , many with hyperbasophilic  c y t o p l a s m (Crowther e t a l . , 1969).  are p r o b a b l y d e r i v e d  from s m a l l o r medium lymphocytes t h a t are  to a n t i g e n i c s t i m u l a t i o n .  into mitosis  t h a t are  allogeneic target c e l l s  and  eventually  small synenter  Lymphocytes have been shown to respond i n  a s i m i l a r manner to a l l o g e n e i c c e l l s basophilic cells  responding  o r mitogens, e n t e r DNA  into large basophilic c e l l s ,  ( L i n g , 1968).  cells  In v i t r o experiments have shown t h a t  lymphocytes upon s t i m u l a t i o n w i t h a n t i g e n t h e s i s , "transform"  These  in vitro,  g i v i n g r i s e to  themselves s p e c i f i c a l l y  (Ginsburg  and  Sachs, 1965;  large  cytotoxic for  the  Ginsburg et a l . ,  110  1969;  G i n s b u r g , 1971; Ax and Koren, 1972).  a n t i g e n presumably f o l l o w s cells  The i n v i v o  a s i m i l a r pattern.  response t o  The a t y p i c a l mononuclear  found i n the b l o o d o f a l l o g r a f t r e c i p i e n t s , t h e r e f o r e ,  represent small  probably  lymphocytes which have been a c t i v a t e d by a l l o g r a f t  a n t i g e n s and which a r e undergoing b l a s t t r a n s f o r m a t i o n and u l t i m a t e l y cell division.  B.  Lymphocyte A c t i v a t i o n S t u d i e s i n +/+ The  uptake o f a g r e a t e r  and W/W  v  Mice  amount o f t r i t i a t e d  t h y m i d i n e by p e r i -  v pheral blood c e l l s  o f W/W  mice b e a r i n g s k i n a l l o g r a f t s than by p e r i -  p h e r a l b l o o d c e l l s o f +/+ greater  mice b e a r i n g a l l o g r a f t s i s good e v i d e n c e  numbers o f a c t i v a t e d  of the mutant a n i m a l s .  lymphocytes a r e p r e s e n t i n the c i r c u l a t i o n  The p r e s e n c e o f g r e a t  numbers o f a c t i v a t e d  lymphocytes i n these mice s u g g e s t s t h a t more c e l l s to g r a f t a n t i g e n s have been s t i m u l a t e d lation.  These r e s u l t s alone a r e s t r o n g  r e a c t i o n o f W/W  V  and  that  and r e l e a s e d  capable of responding i n t o the c i r c u -  evidence that  the a l l o g r a f t  mice i s somewhat more v i g o u r o u s than t h a t  o f +/+  mice,  as such c o n s t i t u t e s u b s t a n t i a l s u p p o r t f o r the r e s u l t s o f e a r l i e r  h i s t o l o g i c a l s t u d i e s which a l s o i n d i c a t e d a more i n t e n s e  graft  reaction  i n the mutant mice. C.  A C o r r e l a t i o n o f O b s e r v a t i o n s from Lymphocyte A c t i v a t i o n and A l l o g r a f t R e j e c t i o n S t u d i e s i n +/+ and W/W Mice  Studies  v  The W/W  c o r r e l a t i o n between a more v i g o u r o u s g r a f t r e a c t i o n i n  mice, as s u g g e s t e d by h i s t o l o g i c a l s t u d i e s ,  activated  cells  a greater  i n the p e r i p h e r a l b l o o d , and a g r e a t e r  number o f  proportion  of  Ill  transformed  cells  is particularly vated c e l l s suggests of  i n the c e l l u l a r  intriguing.  presence V  cells  of  cells  mice b e a r i n g s k i n  allografts  i n the mutant h o s t s , c e l l s which are then r e l e a s e d I f g r e a t e r numbers of  are p r e s e n t i n the p e r i p h e r a l b l o o d ,  sensitized  c e l l s w i l l a l s o be p r e s e n t among the c e l l s  random r a t h e r than s p e c i f i c .  c e l l s may  i n t e r a c t with  infiltrating  Once i n the g r a f t , these  the c e l l s  of the g r a f t and  they p r o b a b l y  lymphokines.  I f g r e a t e r numbers o f s e n s i t i z e d c e l l s  more i n t e n s e . may of  these substances  u n s e n s i t i z e d lymphocytes may  numbers of t a r g e t c e l l s may  of  w i l l be  are present i n be produced,  and  correspondingly  be  be  to MIF  transformed  damaged by  and ECF,  g r e a t e r numbers  by LTF and TF, and  lymphotoxin.  greater  Whether or not  i n f i l t r a t e i s i n d e e d g r e a t e r , i n terms o f a b s o l u t e numbers  c e l l s , i n g r a f t s on W/W  p r o p o r t i o n of t r a n s f o r m e d and  cyto-  Thus, g r e a t e r numbers of macrophages and e o s i n o p h i l s  accumulate i n the g r a f t i n response  the c e l l u l a r  sensitized  g r a f t a n t i g e n s and r e l e a s e  then g r e a t e r q u a n t i t i e s of lymphokines may  the r e a c t i o n s mediated by  grafts  exert a d i r e c t  toxic effect;  the g r a f t  also i n t e r a c t with  acti-  then g r e a t e r numbers  s k i n a l l o g r a f t s , even i f the l o c a l i z a t i o n of c e l l s w i t h i n those is  acti-  a n t i g e n s have succeeded i n a c t i v a t i n g g r e a t e r numbers  from the lymph nodes i n t o the c i r c u l a t i o n . vated  itself  o f g r e a t e r numbers of  i n the p e r i p h e r a l b l o o d of W/W  that g r a f t  lymphoid  The  i n f i l t r a t e w i t h i n the g r a f t  h o s t s , has not been determined.  That  lymphocytes i n the g r a f t s , however, i s g r e a t e r ,  the h i s t o l o g i c a l p i c t u r e somewhat more s e v e r e , and  t h a t these  meters are c o r r e l a t e d w i t h i n c r e a s e d numbers of a c t i v a t e d c e l l s p e r i p h e r a l b l o o d , suggests  the  t h a t the number o f c e l l s  a b l e to  para-  i n the  respond  to  g r a f t antigens  may be a key d i f f e r e n c e between +/+  and may be a s i g n i f i c a n t parameter i n d e t e r m i n i n g  and W/W  mice,  v  the i n t e n s i t y o f the  graft reaction. W/W  V  mice appear t o r e j e c t weakly a n t i g e n i c s k i n g r a f t s i n  l e s s time than do +/+ mice, a l t h o u g h  the r e j e c t i o n o f s t r o n g l y a n t i -  g e n i c g r a f t s does not appear t o be f a s t e r  than normal.  This  differen-  t i a l response to s t r o n g and weak a n t i g e n s  i s perhaps a l s o most  readily  v e x p l a i n e d i n terms o f the numbers o f c e l l s i n +/+ are r e a c t i v e t o s t r o n g and weak g r a f t  and W/W  antigens.  McGregor and Gowans (1964) and Roser and F o r d ported  mice t h a t  (19 72) have r e -  t h a t the s u r v i v a l time o f s k i n a l l o g r a f t s exchanged a c r o s s major  h i s t o c o m p a t i b i l i t y b a r r i e r s i s o n l y m a r g i n a l l y reduced  i n lymphocyto-  p e n i c mice; lymphocytopenia has a more marked e f f e c t , however, when o n l y minor h i s t o c o m p a t i b i l i t y d i f f e r e n c e s e x i s t between donor and h o s t . The  reason  f o r t h e d i f f e r e n t i a l e f f e c t o f lymphocyte d e p l e t i o n on s t r o n g  and weak a l l o g r a f t  r e a c t i o n s i s n o t known.  however, c i t e e v i d e n c e antigens  Roser and F o r d  t h a t the number o f c e l l s  (1972),  reactive with  i s much g r e a t e r than the number o f c e l l s  r e a c t i v e w i t h weak  a n t i g e n s , and s p e c u l a t e t h a t even i n lymphocytopenic animals tration of c e l l s than  t h e concen-  a b l e t o respond to s t r o n g a n t i g e n s may be g r e a t e r  the concentration of c e l l s  animals.  strong  They go on t o suggest  r e a c t i v e t o weak a n t i g e n s  i n untreated  that  above a c r i t i c a l c o n c e n t r a t i o n o f a n t i g e n - s e n s i t i v e c e l l s an i n c r e a s e i n the c o n c e n t r a t i o n o f c e l l s has l i t t l e e f f e c t on s k i n g r a f t s u r v i v a l time (which may be dependent on the m i n i mum time r e q u i r e d t o generate e f f e c t o r c e l l s ) , b u t below t h i s c o n c e n t r a t i o n the s u r v i v a l time depends on the concent r a t i o n of a n t i g e n - s e n s i t i v e c e l l s .  113  R e s u l t s o f a l l o g r a f t r e j e c t i o n i n W/W hypothesis.  A l t h o u g h the r e j e c t i o n o f H-2  mice i s a s s o c i a t e d  v  mice suggest a s i m i l a r  incompatible grafts  w i t h g r e a t e r numbers o f a c t i v a t e d  cells  grafts  picture,  themselves, and w i t h a g e n e r a l l y  the MST o f these g r a f t s  reactive  cells,  critical  no e f f e c t on g r a f t s u r v i v a l times.  the  s u r v i v a l time o f f i r s t - s e t  within  because, as  concentration of  an i n c r e a s e i n the numbers o f r e a c t i v e  has  cells  simply  I t may not be p o s s i b l e  a l l o g r a f t s below about e i g h t  cells.  A l l o g r a f t s b e a r i n g weak anti'gens, however, s u r v i v e  below the c r i t i c a l number r e q u i r e d  o f animals b e a r i n g weakly a n t i g e n i c  of r e a c t i v e  a number  grafts  s u r v i v a l times:  c e l l s between groups  may, t h e r e f o r e ,  have  W/W  V  numbers o f r e a c t i v e  time.  mice may have g r e a t e r numbers o f c e l l s  to respond t o g r a f t a n t i g e n s than do +/+  signi-  an i n c r e a s e i n t h e number  c e l l s might s i g n i f i c a n t l y decrease g r a f t s u r v i v a l  In summary:  antigenic  cells,  t o cause g r a f t r e j e c t i o n i n minimum  i n the c o n c e n t r a t i o n o f r e a c t i v e  f i c a n t e f f e c t s on g r a f t  two t o t h r e e  i n c o m p a t i b l e g r a f t s , perhaps because they  n o r m a l l y s t i m u l a t e a much s m a l l e r number o f r e a c t i v e  Differences  f o r healing,  s e n s i t i z a t i o n , and the p r o d u c t i o n o f e f f e c t o r  times l o n g e r than do H-2  time.  t o reduce  or nine  days, a l i m i t p r o b a b l y dependent on the minimum time r e q u i r e d antigen recognition,  V  more s e v e r e h i s t o l o g i c a l  i s n o t reduced, p o s s i b l y  Roser and F o r d s u g g e s t , beyond a c e r t a i n  W/W  i n the p e r i -  p h e r a l blood, with a g r e a t e r p r o p o r t i o n of transformed c e l l s the  in  able  mice, b u t an i n c r e a s e i n the  c e l l s has no e f f e c t on s u r v i v a l times o f s t r o n g l y  g r a f t s , which n o r m a l l y a c t i v e many more than the c r i t i c a l  number o f a n t i g e n - s e n s i t i v e  cells.  In the case o f weakly  antigenic  114  g r a f t s , however, which n o r m a l l y a c t i v a t e fewer a n t i g e n - s e n s i t i v e and  are rejected slowly,  cells  the p r e s e n c e o f g r e a t e r numbers of r e s p o n s i v e  v c e l l s i n W/W  mice s i g n i f i c a n t l y  III.  A.  General  s h o r t e n s g r a f t s u r v i v a l time.  HUMORAL IMMUNE RESPONSES  Discussion  Lymphocytes have been i m p l i c a t e d i n t h e p r o d u c t i o n  of antibody  a t l e a s t s i n c e 1935 when McMaster and Hudack demonstrated t h a t t h e formation workers  of b a c t e r i a l agglutinins  took p l a c e  i n lymph nodes.  Many  (e.g. Dougherty e t a l . , 1944; H a r r i s eit a_l. , 1945) b e l i e v e d  that the antibody-forming c e l l the g e n e r a l  (AFC) was the s m a l l lymphocyte,  although  consensus h e l d t h a t plasma c e l l s were the a n t i b o d y p r o d u c e r s  (Bjorneboe and Gormsen, 1943; Fagraeus, 1948; Y o f f e y , Leduc et_ a_l. , u s i n g  1964).  the now c l a s s i c f l u o r e s c e n t a n t i b o d y  I n 1955,  technique  of Coons e t a l . (1955), succeeded i n d e m o n s t r a t i n g beyond doubt a n t i b o d i e s were produced by plasma c e l l s .  that  L a t e r s t u d i e s , however,  made i t c l e a r t h a t lymphocytes, t o o , were e s s e n t i a l f o r a n t i b o d y thesis: by  Gowans e t a l . (1962) showed t h a t r a t s d e p l e t e d  chronic  d r a i n a g e o f the t h o r a c i c duct gave a s e v e r e l y  response to a n t i g e n , of s m a l l lymphocytes.  and t h a t t h i s  syn-  o f lymphocytes depressed  response was r e s t o r e d by i n j e c t i o n s  Gowans (1962) a l s o demonstrated t h a t  lymphocytes  could  change i n t o d i v i d i n g c e l l s w i t h new m o r p h o l o g i c a l c h a r a c t e r i s t i c s ,  cells  r e s e m b l i n g those seen i n r e g i o n a l lymph nodes d u r i n g  formation,  and he s u g g e s t e d t h a t the s m a l l  antibody  lymphocyte might be the  115  p r e c u r s o r o f the plasma c e l l .  Other work ( H a r r i s  e t a l , , 1966;  McGregor  et. a l . , 1967; B i r b e c k , 1967) has p r o v i d e d more d e f i n i t i v e e v i d e n c e o f the  t r a n s f o r m a t i o n o f lymphocytes i n t o plasma c e l l s .  c o n s i d e r s i t " l i k e l y , " and R o i t t  et d .  Weiss  (19 72)  now  (1969) c o n s i d e r i t " c l e a r "  t h a t c e r t a i n s m a l l lymphocytes can respond to a n t i g e n by e n l a r g i n g , p r o l i f e r a t i n g , and p r o d u c i n g a p o p u l a t i o n o f a n t i b o d y - f o r m i n g plasma  cells.  Recent s t u d i e s u s i n g s p e c i a l t e c h n i q u e s f o r the l o c a l i z a t i o n of a n t i b o d y by e l e c t r o n  m i c r o s c o p y have demonstrated a n t i b o d i e s i n  both lymphocytes and plasma c e l l s , clearest picture  o f the c e l l s  and have p r e s e n t e d perhaps the  i n v o l v e d i n antibody s y n t h e s i s .  Antibody  appears to be formed i n t y p i c a l plasma c e l l s , i n some l a r g e lymphocytes, and i n c e r t a i n  cells  ("lymphoplasmacytes") which have some m o r p h o l o g i c a l  c h a r a c t e r i s t i c s o f b o t h plasma c e l l s and s m a l l lymphocytes  (Avrameas  and B o u t e i l l e , 1968; Leduc e t a l . , 1968; Avrameas and Leduc, 1970). Lymphocytes, a l t h o u g h m o r p h o l o g i c a l l y homogeneous, a r e known to comprise a t l e a s t two d i s t i n c t s u b p o p u l a t i o n s , a  thymus-dependent  p o p u l a t i o n and a thymus-independent p o p u l a t i o n ; these a r e d e s i g n a t e d respectively  T - c e l l s and B - c e l l s  (see R o i t t  e t a l . , 1969; R a f f ,  and c a r r y c h a r a c t e r i s t i c s u r f a c e a n t i g e n markers. sible  1970)  Those c e l l s r e s p o n -  f o r the p r o d u c t i o n o f humoral a n t i b o d i e s b e l o n g to the B - c e l l  population  ( f o r review see S t r o b e r , 19 75).  Experiments summarized by Weiss some s o r t  of c o o p e r a t i o n between  (1972) have demonstrated t h a t  lymphocytes and macrophages  o r between  d i f f e r e n t p o p u l a t i o n s o f lymphocytes i s n e c e s s a r y f o r the p r o d u c t i o n of a n t i b o d i e s to c e r t a i n a n t i g e n s .  Antibody-forming B - c e l l s  seem t o  116  require some sort of stimulation from macrophages or from antigensensitive T-cells.  Feldmann (19 73) and Byrd et a l . (19 74) have noted  that those antigens which are T - c e l l dependent are generally monomeric antigens, and have suggested that the role of T - c e l l s and macrophages in the antibody response i s to bind these antigen molecules and present them to B-cells i n a l o c a l l y concentrated form.  There i s evidence that  T - c e l l s produce a soluble immunoglobulin-like factor which binds antigen and i s c y t o p h i l i c for macrophages, rendering them capable of s p e c i f i c a l l y immunizing  B-cells  (Feldmann, 1973).  This model, however, i s  speculative, and the nature of c e l l cooperation i n the antibody response s t i l l controversial and f a r from c l e a r . Other c e l l u l a r events i n the process of antibody synthesis are also s t i l l f a r from being understood.  For some time, however, i t  has been taken for granted that immunoglobulin  receptors on B lympho-  cytes are involved i n antigen recognition by these c e l l s , and i n Bc e l l a c t i v a t i o n (Greaves, 19 70; Nossal et a l . , 19 72; Hammerling and McDevitt, 1974a, 1974b; Kreth and Herzenberg, 1974).  Further, i t i s  generally thought that i n the i n i t i a l or primary response to an antigen such as SRBC, i n t e r a c t i o n with antigen causes a small proportion of s p e c i f i c a l l y reactive c e l l s i n lymphoid organs to undergo c l o n a l expansion, increasing i n number perhaps 1,000  times and giving r i s e to c e l l s  which secrete s p e c i f i c antibodies, most of which are IgM (Shearer et a l . , 1968; Wilson and Nowell, 1971).  Upon second exposure to antigen, the  greater proportion of antigen-reactive c e l l s which now  exists i s be-  lieved to r e s u l t i n the prompt and prolonged production of even higher  117  t i t e r s o f a n t i b o d y , a l t h o u g h the a n t i b o d y now (Wilson and Nowell,  19 71).  production of d i f f e r e n t a n t i b o d y o f o n l y one  lysis  type.  class,  and those capable o f  are p r o b a b l y o f the IgG  the  secreting  class  agglutination  (Wortis e_t a l . , 1966).  types o f a n t i b o d y a r i s e from common  ( S t e r z l and N o r d i n , 19 71) o r from u n i p o t e n t p r e c u r s o r s  (Shearer e t a l . , 1968, I n d i v i d u a l AFC whereby s p l e e n c e l l s  1969a, 1969b) i s u n c e r t a i n . can be d e t e c t e d by  the plaque  formation technique,  are mixed w i t h SRBC and p l a t e d e i t h e r i n agar o r  i n s u s p e n s i o n between g l a s s s l i d e s . AFC  IgG  A n t i b o d i e s capable o f l y s i n g SRBC d i r e c t l y  Whether c e l l s p r o d u c i n g d i f f e r e n t precursors  a r e c h a r a c t e r i z e d by  types of a n t i b o d y , i n d i v i d u a l c e l l s  are p r o b a b l y o f the IgM or i n d i r e c t  I n d i v i d u a l AFC  produced i s l a r g e l y  Target c e l l s surrounding  individual  are l y s e d w i t h i n an hour o r so by a n t i b o d y s e c r e t e d by a c t i v e  cells.  Cunningham (1968b) reports- a v a r i e t y o f m o r p h o l o g i c a l types among mouse AFC  d e t e c t e d by the p l a q u e  account  technique:  f o r a s m a l l number o f p l a q u e s , b u t most AFC  mononuclears w i t h r e l a t i v e l y no and  c l a s s i c a l mature plasma  little  cytoplasm."  plaque  t e c h n i q u e , and  The  "basophilic  Cunningham d i s c e r n s  c o n s i s t e n t m o r p h o l o g i c a l d i f f e r e n c e between c e l l s those r e l e a s i n g IgG.  are  cells  a b s o l u t e number o f AFC  releasing  IgM  d e t e c t e d by  the  the time o f peak response v a r i e s w i t h the dose  of a n t i g e n and the r o u t e o f a d m i n i s t r a t i o n (Cunningham, 1968b; W o r t i s e_t a l . , 1966).  I n t r a p e r i t o n e a l a d m i n i s t r a t i o n r e s u l t s i n a peak  o f d i r e c t plaques by  the f o u r t h day  (Wortis ejt a l . , 1966) .  response  118  B.  Humoral Immune Responses i n +/+ and W/W  V  Mice  The number of spleen c e l l s a c t i v e l y secreting SRBC hemolysins i n W/W  V  mice during the primary response to antigen i s about 1/4 that  in +/+ mice, as determined by plaque assay. agglutinins produced by W/W  V  to that produced by +/+ mice.  The concentration of SRBC  mice, however, i s e s s e n t i a l l y i d e n t i c a l W/W  V  mice might appear, therefore, to  be defective i n hemolysin (IgM) production but not i n hemagglutinin (IgG) production.  This d i f f e r e n t i a l defect, however, may not r e f l e c t  differences i n immunoglobulin  concentration so much as differences  in the s e n s i t i v i t y of the assays used.  A small difference i n agglutinin  concentration would not be detected by the s e r i a l d i l u t i o n technique which, at best, i s subject to a 2-fold error.  In the secondary response  to antigen a small but s i g n i f i c a n t difference between antibody t i t e r s of +/+ and W/W  V  mice indicates that hemagglutinin production i n the  mutant mice i s indeed defective. A defect i n PFC numbers i n W mutant mice has been reported before:  Shearer and Cudkowicz (196 7) found that W /W mice produced V  only 1/3 to 1/2 the number of PFC as did t h e i r W /+ V  V  l i t t e r m a t e s ; this  r a t i o agrees w e l l with the results reported i n this thesis. study, however, finds no difference between +/+ and W/W  V  Another  mice i n the  PFC response to SRBC (Mekori and P h i l l i p s , 1969); indeed, i n this study a s l i g h t l y higher PFC response was found among W mutants than among coisogenic normal mice. An explanation f o r the lack of agreement among the results from d i f f e r e n t laboratories i s not readily apparent.  Mekori and  119  Phillips  used W/W  V  gous f o r W ; V  mice w h i l e S h e a r e r and Cudkowicz used mice homozy-  Mekori and P h i l l i p s  combination o f a l l e l e s may  s u g g e s t , t h e r e f o r e , that the  account  f o r the d i f f e r e n t  different  results.  This  v hypothesis, in  this  however, does not appear  to h o l d :  W/W  mice,  as  reported  t h e s i s , as w e l l as by McMaster (1973), can a l s o produce  t i v e PFC  response.  the experiments  a  defec-  There i s , t h e r e f o r e , no obvious d i f f e r e n c e between  conducted i n the d i f f e r e n t l a b o r a t o r i e s which  might  account f o r the d i s p a r i t y i n r e s u l t s . A number o f mechanisms can be s u g g e s t e d to e x p l a i n the t i v e response o f W/W  V  mice t o SRBC  defec-  antigens:  1) reduced numbers of a n t i g e n - r e a c t i v e  B-cells;  2) a d e f e c t i n the a c t i v i t y o f the h e l p e r c e l l s , e i t h e r T - c e l l s or macrophages, t h a t are n e c e s s a r y f o r a normal response to SRBC; 3) a d e f e c t i n the i n t e r a c t i o n between the B - c e l l and the a n t i g e n , such t h a t a c t i v a t i o n o f B - c e l l s i s i n h i b i t e d ; 4) a d e f e c t i n the a c t i v a t i o n mechanism i t s e l f , fewer B - c e l l become a c t i v a t e d ;  such  5) a d e f e c t i n p r o l i f e r a t i o n , such t h a t fewer AFC produced; 6) d e f e c t i v e immunoglobulin In view o f what appears allograft  response, a h e l p e r  V  production.  c e l l d e f e c t seems perhaps  V  mice.  This  the l e a s t  likely  Shearer and Cudkowicz (1967), however, have  t h a t s p l e e n s o f homozygous W  number o f a n t i g e n - r e a c t i v e as do W /+  are  to be a t l e a s t normal T - c e l l f u n c t i o n i n the  of the above hypotheses. reported  that  cells  (ARC)  mice c o n t a i n about h a l f the (as' determined by focus assay)  r e p o r t suggests t h a t those ARC  which  are a c t i v a t e d  v . v i n W /W mice p r o l i f e r a t e n o r m a l l y and produce normal q u a n t i t i e s of  120  immunoglobulins.  H y p o t h e s i s 1) above appears, t h e r e f o r e , to be  p o r t e d , and hypotheses of ARC  may  5) and 6) to be u n l i k e l y .  be due to the absence  of the c e l l s p r e s e n t to r e a c t 3) and 4).  of these c e l l s  sup-  A deficiency or to the  i n numbers  inability  to s t i m u l a t i o n , as s u g g e s t e d by  hypotheses  P r e s e n t knowledge does n o t a l l o w a c h o i c e between these  p o s s i b i l i t i e s , b u t h y p o t h e s i s 3) w i l l be d i s c u s s e d i n more d e t a i l  IV.  A.  MITOGEN STIMULATION STUDIES  General D i s c u s s i o n S m a l l lymphocytes  m i t o g e n i c agents  can be i n d u c e d by  treatment w i t h v a r i o u s  to undergo c e r t a i n m e t a b o l i c and.morphologic  u l t i m a t e l y r e s u l t i n g i n the f o r m a t i o n of b l a s t - l i k e t i o n was  first  r e p o r t e d by N o w e l l  l e c t i n phytohaemagglutinin b l o o d lymphocytes. and pokeweed mitogen  (PHA)  (1960), who  cells.  1964;  changes,  This reac-  found t h a t the p l a n t  i n d u c e d t r a n s f o r m a t i o n of p e r i p h e r a l  Other p l a n t l e c t i n s such as c o n c a n a v a l i n - A (Con-A) (PWM), and agents such as b a c t e r i a l  (LPS), have s i n c e been r e p o r t e d to have s i m i l a r e f f e c t s P o w e l l and Leon,  endotoxins (Fames et a l . ,  1970).  The i n d u c t i o n o f b l a s t  t r a n s f o r m a t i o n by n o n s p e c i f i c  mitogens  i s not an immunological r e s p o n s e , but i s o f p a r t i c u l a r i n t e r e s t it  later.  appears  to mimic the response o f lymphocytes  c y t e s from u n s e n s i t i z e d i n d i v i d u a l s , p r e s e n c e o f a l l o g e n e i c lymphocytes formation  ( B a i n e_t al. , 1963,  to a n t i g e n .  f o r example, w i l l  Lympho-  respond to the  iti v i t r o by undergoing b l a s t  1964).  Lymphocytes w i l l  because  also  trans-  respond  121  i n v i t r o to other types of c e l l s bearing foreign transplantation a n t i gens, such as embryonic c e l l monolayers (Ax and Koren, 1972; Ginsburg et a l , , 1969; Altman ejt _al. , 1973), and c e l l s of lymphoid l i n e s (Hardy et a l . , 1969).  In addition, lymphocytes from s e n s i t i z e d i n d i v i d u a l s  w i l l undergo marked•transformation and p r o l i f e r a t i o n i f exposed i n v i t r o to the s e n s i t i z i n g antigen ( M i l l s , 1966).  The b l a s t c e l l s a r i s i n g  from unsensitized lymphocytes responding to allogeneic c e l l s , and from s e n s i t i z e d lymphocytes cultured with s p e c i f i c antigens are, s u p e r f i c i a l l y at l e a s t , s i m i l a r to those b l a s t c e l l s a r i s i n g from lymphocytes stimulated n o n - s p e c i f i c a l l y with plant l e c t i n s .  In addition, these  blast c e l l s closely resemble those transformed or activated lymphocytes which appear i n the lymph and peripheral blood of animals bearing a l l o grafts (p.109).  Nonspecific mitogenic stimulation, therefore, i s gener-  a l l y considered not only a model useful i n analyzing the events i n volved i n lymphocyte a c t i v a t i o n , but also a means f o r monitoring the immunological competence of lymphocytes from test subjects (Janossy and Greaves, 19 71). The morphological and u l t r a s t r u c t u r a l changes occurring a f t e r mitogenic stimulation have been described i n d e t a i l (Ling, 1968).  Briefly,  these involve a marked increase i n the nuclear-cytoplasmic r a t i o , the appearance of a highly developed Golgi apparatus and more abundant mitochondria, and increased cytoplasmic basophilia related to increased numbers of ribosomes.  RNA synthesis, protein synthesis, and eventually  DNA synthesis occur, and a f t e r two or three days of treatment mitoses occur.  122  B l a s t c e l l s produced upon lymphocyte t r a n s f o r m a t i o n appear to be c y t o t o x i c f o r t a r g e t c e l l s .  The b l a s t  c u l t u r e d on monolayers o f f o r e i g n c e l l s 1971; G i n s b u r g and Sachs, 1965; histocompatibility  antigens  cells  formed from lymphocytes  (Ax and Koren, 1972; G i n s b u r g ,  Ginsburg et a l . ,  1969), w i t h s u b c e l l u l a r  (Koldovsky e_t a l . , 1969; Manson and Simmons,  1969), o r i n some mixed lymphocyte  cultures  (Andersson and Hayry,  Hodes and Svedmyr, 19 70) a r e r e p o r t e d to be s p e c i f i c a l l y c e l l s o f the s e n s i t i z i n g genotype.  Activated cells  1973;  cytotoxic for  arising  from lympho-  c y t e s s t i m u l a t e d w i t h p l a n t l e c t i n s a r e a l s o r e p o r t e d t o be  cytotoxic  for target c e l l s  1973), b u t  Perlmann et a l . ,  (Holm and Perlmann, 1965;  and Holm (1969) and o t h e r s 1974)  emphasize  Stejskal et a l , ,  (e.g. Asherson e t a l . ,  1973; Warnatz  that this c y t o t o x i c i t y i s nonspecific.  There i s some q u e s t i o n as t o whether the b l a s t c e l l s produced upon mitogen s t i m u l a t i o n and the c y t o t o x i c e f f e c t o r c e l l s c u l t u r e s a r i s e from the same p o p u l a t i o n o f lymphocytes 1972; D a g u i l l a r d , of c y t o t o x i c i t y  1972).  seen i n t h e s e  ( S t i t e s et a l . ,  A l t h o u g h degree o f s t i m u l a t i o n and degree  usually correlate well,  themselves be the e f f e c t o r c e l l s  the t r a n s f o r m e d c e l l s may  ( S t e j s k a l e_t ail. , 1973).  not  Indeed,  r e c e n t r e p o r t s i n d i c a t e t h a t the g e n e r a t i o n of c y t o t o x i c e f f e c t o r  lympho-  c y t e s need n o t c o r r e l a t e a t a l l w i t h mitogen-induced t r a n s f o r m a t i o n o r w i t h mixed lymphocyte r e a c t i v i t y , and i s i n f a c t independent o f the p r o l i f e r a t i v e r e s p o n s e ( R o c k l i n , 1972; Mawas e_t al. , 1975;  C o r l e y e_t.  a l . , 19 75). The h y p o t h e s i s t h a t a n t i g e n s a c t i v a t e lymphocytes v i a r e c e p t o r s on the lymphocyte s u r f a c e i s now  g e n e r a l l y a c c e p t e d (see D i e n e r and  123  Langman, 1975,  f o r review).  s p e c i f i c surface  receptors  Normal mouse lymphocytes appear to f o r antigens  i n c l u d i n g h i s t o c o m p a t i b i l i t y antigens  (Hammerling and  are immunoglobulins 1972;  (Melchers and  M a r c h a l o n i s and  19 74; V i t e t t a e t a l . , 19 71);  Consider-  at l e a s t i n B - c e l l s ,  Andersson, 1973;  Cone, 1973;  M c D e v i t t , 19 74),  (Altman e t a l , , 1973).  a b l e e v i d e n c e i n d i c a t e s t h a t these r e c e p t o r s ,  carry  M a r c h a l o n i s et a l . ,  N o s s a l e_t a l . , 1972;  Pernis  et a l . ,  the n a t u r e of the T - c e l l r e c e p t o r  is  still  controversial. N o n s p e c i f i c mitogens such as to a c t i v a t e lymphocytes v i a s u r f a c e to l o c a l i z e w i t h i n 1973), but  cells  the p l a n t l e c t i n s a l s o appear receptors.  ( S t a n l e y e t a l , , 1969;  PHA  has  been  Rubin and  the i n i t i a l event a t l e a s t i n v o l v e s a membrane  reported  Schultz, receptor.  Polgar  e_t al_. (1969) r e p o r t a d i r e c t r e l a t i o n s h i p between the  of PHA  to c e l l s  and  the m e t a b o l i c a l t e r a t i o n s which f o l l o w , and  e t a l , (1971) r e p o r t t h a t the m i t o g e n i c a c t i v i t y tively The  of PHA  adsorbed by plasma membranes of lymphocytes and  n a t u r e o f the PHA  dered most l i k e l y  r e c e p t o r has  to be  can be thymus  not been determined, but  a glycoprotein  f o r the a n t i g e n i c d e t e r m i n a n t s of LPS,  LPS  the  receptors  Con-A, on  responsible  van  den  cells.  i s consi-  receptors  Berg, 1972).  The  these  for nonspecific activation, (Andersson e t a l . ,  the o t h e r hand, appears to b i n d s e l e c t i v e l y  alpha-D-mannoside i n the lymphocyte s u r f a c e B e t e l and  effec-  evidence i n d i c a t e s that  r e c e p t o r s , however, have not been c h a r a c t e r i z e d  1972a).  Allan  ( D a g u i l l a r d , 1972).  A l t h o u g h lymphocytes c a r r y s p e c i f i c immunoglobulin  are u n l i k e l y to be  binding  (Powell  and  to methyl  Leon, 19 70;  a d d i t i o n of these sugars to  the  124  culture system reduces the binding of Con-A to the c e l l , but does not a f f e c t the binding of PHA  (Powell and Leon, 19 70).  The i n i t i a l event  in lymphocyte a c t i v a t i o n by mitogen, therefore, appears to involve surface receptors, and these receptors are probably  mitogen-specific.  The mechanism by which non-specific mitogens activate, lymphoid c e l l s i s not known.  Although PHA has been reported to enter the  and bind to heterochromatin  (Rubin and Schultz, 1973;  1969), most reports i n d i c a t e that prolonged  nucleus  Stanley et a l . ,  i n t e r a c t i o n of mitogen with  the c e l l surface receptor i s s u f f i c i e n t to induce transformation (Betel and van den Berg, 1972),  A c t i v a t i o n of chromatin, then, may  occur  through one or several secondary messengers (Skoog et a l . , 19 73) possibly involving cyclic-AMP  (Krishnaraj and Talwar, 19 73).  PHA  and Con-A  are reported to cause as much as a 3-fold increase i n cAMP l e v e l s i n lymphocytes within one minute of incubation (Parker, 1974).  Indeed,  Watts (19 71) has proposed that the a c t i v a t i o n of c e l l s by antigen  may  be analogous to the action of hormones on c e l l s , i n v o l v i n g cAMP as a secondary messenger.  Although there i s thus reason to suspect that  c y c l i c nucleotides are involved i n lymphocyte a c t i v a t i o n , t h e i r role i s not yet firmly established, and the mechanism of mitogen a c t i v a t i o n remains unclear. D i f f e r e n t mitogens appear to activate d i f f e r e n t of small lymphocytes.  In mice, for example, PHA  populations  and Con-A appear to  be s e l e c t i v e l y active on T - c e l l s , and LPS s e l e c t i v e l y active on B - c e l l s (Andersson et a l . , 1972a; Peavy et a l . , 1974; 1971).  Janossy and Greaves,  The basis for this s e l e c t i v e a c t i v a t i o n , however, i s not known.  125  The  p r e s e n c e or absence of a p p r o p r i a t e  some i n s t a n c e s , but  i n the  receptors  be  relevant i n  case of Con-A, f o r example, B-  appear to c a r r y e q u a l numbers of r e c e p t o r s same e x t e n t  may  ( M o l l e r e t a l . , 19 73).  and  and  to b i n d Con-A to  B - c e l l a c t i v a t i o n by  t h e r e f o r e , to r e q u i r e more than b i n d i n g  T-cells  Con-A appears,  o f Con-A to the s u r f a c e .  have been proposed to e x p l a i n s e l e c t i v e a c t i v a t i o n i n terms of cooperation  the  Models  cell  or requirements f o r s o l u b l e s u b s t a n c e s a f t e r b i n d i n g  taken p l a c e , but  has  these are s t i l l h i g h l y s p e c u l a t i v e and c o n t r o v e r s i a l  (Andersson, e t a l . , 1972a, 19 72b).  B.  Mitogen S t i m u l a t i o n S t u d i e s The  LPS  response of s p l e e n  is clearly  i n d i c e s o f W/W  V  regardless  deficient:  i n +/+ cells  b o t h raw  and W/W  Mice  V  from W/W  mice to the  count means and mean s t i m u l a t i o n  c u l t u r e s are always lower than those of +/+  o f the c o n c e n t r a t i o n  mitogen  cultures,  of mitogen used o r the l e n g t h o f  i n c u l t u r e (the d e f i c i e n c y , however, i s most marked a t l o n g e r periods).  A number o f hypotheses may  be  time  culture  proposed to e x p l a i n the  defi-  v c i e n t response o f W/W  spleen  cells  to  LPS:  1. a s m a l l e r p r o p o r t i o n of B - c e l l s and t h e r e f o r e a s m a l l e r p r o p o r t i o n of L P S - r e s p o n s i v e c e l l s i n W/W cultures; v  2.  a d e f i c i e n c y of LPS r e c e p t o r s on W/W c e l l s w i t h a consequent d e f i c i e n c y i n the b i n d i n g of LPS to W/Wv cells; V  3. i n a b i l i t y cells. Present  o f bound LPS  knowledge does not  to s t i m u l a t e  allow  transformation  a c h o i c e between these  in  W/W  V  possibilities.  v The  response of s p l e e n  cells  from W/W  C o n c a n a v a l i n - A appears to be m a r g i n a l l y  mice to the p l a n t  stronger  than the  lectin  response  126  of +/+  spleen c e l l s  of W/W  V  cells  c e l l s , but, i n W/W  V  to t h i s mitogen.  Stimulation indices for cultures  are not a t a l l d i f f e r e n t  i f the raw  counts  from those  f o r the two  f o r c u l t u r e s of  samples are compared,  c u l t u r e s appear h i g h e r at 72 hours than do counts  t u r e s , and a t 96 hours, when the o p t i m a l response this  difference is s t a t i s t i c a l l y  significant.  be p r o p o s e d to e x p l a i n the enhanced response  counts  i n +/+  t o Con-A i s  +/+  cul-  reached,  S e v e r a l hypotheses of W/W  v  cells  may  to Con-A:  1. a g r e a t e r p r o p o r t i o n of T - c e l l s and t h e r e f o r e a g r e a t e r p r o p o r t i o n of Con-A r e s p o n s i v e c e l l s i n W/Wv cultures; 2.  some a l t e r a t i o n i n the s u r f a c e c h a r a c t e r of W/W cells which r e s u l t s i n o p t i m a l b i n d i n g of the l e c t i n and thereby optimal s t i m u l a t i o n ; V  3. i n c r e a s e d s e n s i t i v i t y transformation.  of W/W  V  Con-A b i n d i n g c e l l s  P r e s e n t knowledge does not a l l o w a c h o i c e between these The depressed  c o r r e l a t i o n between depressed  responses  i n B - c e l l responses. rejection  to LPS  i n W/W  V  Likewise,  antibody  mice suggests  to  possibilities.  production  and  a general deficiency  the c o r r e l a t i o n between enhanced g r a f t  and p o s s i b l y enhanced responses  a g e n e r a l enhancement of T - c e l l r e s p o n s e s .  to Con-A i n t h e s e mice Further studies using  suggests other  T - c e l l and B - c e l l mitogens and o t h e r T - c e l l and B - c e l l a n t i g e n s would be  r e q u i r e d to determine whether or not  fact, hold. suggests later.  The  possibility  t h i s g e n e r a l i z a t i o n does, i n  of a l t e r e d lymphocyte r e a c t i v i t y , however,  an i n t r i g u i n g h y p o t h e s i s which w i l l be  d i s c u s s e d i n more  detail  127  V.  A.  Macrophage M i g r a t i o n  OTHER STUDIES  Studies  The o b s e r v a t i o n t h a t p r i m o r d i a l germ c e l l s  i n W/W  v  embryos v  are s e v e r e l y r e t a r d e d i n m i g r a t i o n , and t h a t lymphocytes  from  W/W  mice a l s o m i g r a t e more s l o w l y than normal, s u g g e s t e d t h a t o t h e r m i g r a tory c e l l s  i n these mice might be a f f e c t e d i n a s i m i l a r manner.  The m i g r a t i o n o f macrophages from c a p i l l a r y and the i n h i b i t i o n  tubes i n c u l t u r e ,  of t h i s m i g r a t i o n by the p r e s e n c e o f s e n s i t i z e d  lymphocytes and a n t i g e n , has o f t e n been used as an i n v i t r o of c e l l u l a r h y p e r s e n s i t i v i t y The  capillary  (see Friedman e t a l . ,  for references).  tube c u l t u r e system seemed t o o f f e r a s i m p l e method o f  d e t e r m i n i n g whether phage m i g r a t i o n .  o r not t h e W gene e x e r t s any g r o s s e f f e c t on macro-  Experiments demonstrated no d i f f e r e n c e i n m i g r a t i o n  between p e r i t o n e a l exudate c e l l s capillary  1969,  correlate  from +/+  and from W/W  V  mice.  tube c u l t u r e method, however, proved to be h i g h l y  The  variable  and n o t , t h e r e f o r e , p a r t i c u l a r l y s e n s i t i v e ; s m a l l but s i g n i f i c a n t f e r e n c e s i n m i g r a t i o n between e x p e r i m e n t a l and c o n t r o l groups t h e r e f o r e , not be d e t e c t e d by t h i s p r o c e d u r e .  would,  These experiments  o n l y t o the c o n c l u s i o n t h a t the W gene e x e r t s no g r o s s e f f e c t  dif-  lead  on macro-  phage m i g r a t i o n .  B.  Lymphocyte A d h e s i o n S t u d i e s . Lymphocytes  b l a s t s , and  are n o t a d h e s i v e i n the way  o t h e r c e l l s which  t h a t macrophages,  grow i n c u l t u r e a r e .  fibro-  T h i s l a c k o f adhe-  s i v e n e s s a l l o w s them to be e a s i l y s e p a r a t e d from such c e l l s  as macrophages  128 simply by  a l l o w i n g a mixed c e l l p o p u l a t i o n  an hour; macrophages w i l l adhere f i r m l y c y t e s can be washed away. in  to s e t t l e on g l a s s f o r h a l f  to the g l a s s and  the  lympho-  Lymphocytes, a r e , however, somewhat  "sticky"  t h a t a c e r t a i n p e r c e n t a g e of them w i l l adhere g e n t l y to g l a s s  will  remain a t t a c h e d  against  f o r c e of g r a v i t y u n l e s s  the  and  f o r c e f u l l y  washed away. The  ability  of c e l l s  to adhere to s u r f a c e s  i s one  very  method o f i n v e s t i g a t i n g c e r t a i n c e l l s u r f a c e p r o p e r t i e s . of the c e l l s  The  simple treatment  themselves, or o f the s u r f a c e s on which they w i l l be  w i t h v a r i o u s a g e n t s , determines the p r o p o r t i o n of adherent c e l l s helps  to i n d i c a t e what f a c t o r s i n f l u e n c e c e l l a d h e s i o n .  though l i m i t e d i n what they  can  Such s t u d i e s ,  cells.  C e l l s must adhere to s u r f a c e s i n o r d e r t o m i g r a t e ,  f o r e , t h a t the r e t a r d e d m i g r a t i o n i n W/W  V  mice may  be  The  s p e c u l a t i o n was  of lymphocytes and  associated with  s u r f a c e , such t h a t a d h e s i o n ,  and  weak a d h e s i o n of lymphocytes  to g l a s s was  determining populations.  and  i t seems  assumption t h a t i f a d h e s i o n i s e i t h e r too g r e a t o r  weak, m i g r a t i o n w i l l be h i n d e r e d .  cells  thereby  T e s t s showed, however, no cells  advanced,  too there-  c e r t a i n other  some a l t e r a t i o n i n the migration,  cell  i s affected.  used as a s i m p l e  the degree of a d h e s i v e n e s s of +/+  p o r t i o n s of a d h e s i v e  and  demonstrate, do p r o v i d e a c e r t a i n amount  of i n f o r m a t i o n about the s u r f a c e p r o p e r t i e s of  a reasonable  placed,  and W/W  V  method of  spleen  cell  d i f f e r e n c e s between the  i n mutant and non-mutant mice.  The  pro-  129 VI.  No  HAEMATOLOGY  d i f f e r e n c e s a r e apparent between +/+  mice and mice  carrying  the W mutation w i t h r e g a r d t o t o t a l and d i f f e r e n t i a l w h i t e b l o o d c o u n t s , e i t h e r b e f o r e or a f t e r g r a f t i n g .  A d u l t W/W  by C h e r v e n i c k and Boggs (1969), a r e c l e a r l y normal numbers o f c i r c u l a t i n g  mice, as  V  cell  reported  capable o f m a i n t a i n i n g  g r a n u l o c y t e s and lymphocytes, and  appear  to respond to t r a n s p l a n t a t i o n p r o c e d u r e s i n the same manner as do mice.  The  following discussion w i l l  the genotypes  o f the mice  t h e r e f o r e make no r e f e r e n c e t o  involved.  Mice b e a r i n g s k i n a u t o g r a f t s o r a l l o g r a f t s depressed white c e l l  counts.  Striking variability  i n mice i s not u n u s u a l , and the changes p l a n t a t i o n probably r e f l e c t ,  at l e a s t  t h e s i a and the g e n e r a l trauma p l a n t a t i o n process i t s e l f . differential in  i n white c e l l  associated with skin  the a l l o g r a f t .  trans-  d e m o n s t r a t i n g a marked d e c l i n e The lymphocytopenia a s s o -  r e j e c t i o n has n e v e r been f u l l y  e x p l a i n e d , but lymphocytes  The o b s e r v a t i o n t h a t lymphocytopenia i s g r e a t e s t  a f t e r day s i x o r seven, when the c e l l u l a r i n f i l t r a t e is  count  In mice b e a r i n g s k i n a l l o g r a f t s , however,  be a r e f l e c t i o n o f the movement o f l a r g e numbers o f  into  markedly  i n p a r t , the e f f e c t s o f anaes-  the r a t i o of c i r c u l a t i n g lymphocytes.  may  demonstrate  r e s u l t i n g from h a n d l i n g and the t r a n s -  counts a r e a l s o changed,  ciated with allograft  normal  i n the  r e a c h i n g a peak, i s c o n s i s t e n t w i t h t h i s h y p o t h e s i s .  allograft  130 VII.  SPECULATION: THE PRIMARY SITE OF ACTION OF THE W GENE  L e t me now  summarize the r e s u l t s o f t h i s study of immune  v responses i n W/W  mice,  and s p e c u l a t e on an e x p l a n a t i o n f o r these  r e s u l t s and on a h y p o t h e s i s f o r the primary s i t e of a c t i o n of the W gene. With r e g a r d to immune responses i n W/W 1) weakly  that:  a n t i g e n i c s k i n g r a f t s are r e j e c t e d more q u i c k l y by  mice than by +/+  mice.  G r a f t s b e a r i n g s t r o n g a n t i g e n s are n o t  more q u i c k l y b u t e l i c i t  a somewhat more i n t e n s e inflammatory  that i s detectable h i s t o l o g i c a l l y 2) g r a f t  mice, i t appears  r e j e c t i o n i n W/W  V  rejected  response period;  mice i s a s s o c i a t e d w i t h g r e a t e r numbers  V  of a c t i v a t e d c e l l s  d u r i n g the a c u t e r e j e c t i o n  W/W  i n the p e r i p h e r a l b l o o d , and, a c c o r d i n g to C o l l e n  (1974), w i t h a l a r g e r p r o p o r t i o n o f t r a n s f o r m e d lymphocytes w i t h i n the g r a f t  itself;  3) s p l e e n c e l l s  from W/W  V  mice produce  i n response to SRBC than do s p l e e n c e l l s 4) s p l e e n c e l l s  from W/W  V  fewer p l a q u e - f o r m i n g  from +/+  cells  mice;  mice e x h i b i t d e p r e s s e d B - c e l l  responses  and, p o s s i b l y , m a r g i n a l l y enhanced T - c e l l responses when t e s t e d w i t h B - c e l l and T - c e l l  mitogens.  O b s e r v a t i o n s 1 and 2 are most r e a d i l y  e x p l a i n e d by  the hypo-  v t h e s i s t h a t W/W  mice c o n t a i n g r e a t e r numbers of c e l l s  g r a f t a n t i g e n s than do +/+ mice c o n t a i n fewer c e l l s mice.  S i n c e the g r a f t  mice.  reactive with  O b s e r v a t i o n 3 suggests t h a t  W/W  V  a b l e to respond t o SRBC a n t i g e n s than do  r e a c t i o n i s p r i m a r i l y a T - c e l l response,  +/+  and  131 antibody  production  a B - c e l l response, these  observations  l e a d t o the  s p e c u l a t i o n t h a t T - c e l l responses g e n e r a l l y may be somewhat enhanced / v i n W/W  mice and B - c e l l responses depressed.  some support  from o b s e r v a t i o n  to B - c e l l mitogens i n W/W  T h i s s p e c u l a t i o n gaxns  4 , which i n d i c a t e s t h a t the response  mice i s depressed and the response t o T - c e l l  V  mitogens perhaps m a r g i n a l l y  enhanced.  Enhanced T - c e l l responses and d e p r e s s e d B - c e l l responses may be  e x p l a i n e d by p o s t u l a t i n g the e x i s t e n c e o f an a l t e r e d T - c e l l / B - c e l l  r a t i o i n W/W  V  mice:  perhaps the p r o p o r t i o n o f T - c e l l s i s h i g h e r and  the p r o p o r t i o n o f B - c e l l s lower i n these mice than i n normal mice. An  attempt t o determine the T - c e l l / B - c e l l r a t i o i n the s p l e e n s  and W/W  V  mice by l a b e l l i n g B - c e l l s w i t h  fluorescein-conjugated  o f +/+ anti-  immunoglobulin, however, has n o t y e t y i e l d e d r e s u l t s . An is  a l t e r n a t i v e explanation  t o an a l t e r e d T - c e l l / B - c e l l  t h a t normal numbers o f T- and B - c e l l s i n W/W  V  i n t h e i r r e a c t i v i t y with  leads  to a hypothesis  mice a r e s i m p l y a l t e r e d  antigens, T - c e l l r e a c t i v i t y being  and B - c e l l r e a c t i v i t y b e i n g  depressed.  ratio  S p e c u l a t i o n on t h i s  enhanced possibility  which may be a b l e to e x p l a i n n o t o n l y t h e anoma-  l o u s immune responses o f W mice, b u t a l l the o t h e r known e f f e c t s o f the W gene as w e l l . The believed with  response o f lymphocytes  to a n t i g e n s  to be i n i t i a t e d by the i n t e r a c t i o n of the s t i m u l a t i n g m o l e c u l e  a r e c e p t o r on the lymphocyte s u r f a c e .  poiesis  and t o mitogens i s  E r y t h r o p o i e s i s and l e u k o -  a r e r e g u l a t e d by hormones which a r e a l s o known t o e x e r t  effects v i a c e l l surface receptors.  P r i m o r d i a l germ c e l l s -and  their  132  prospective  pigment c e l l s may  perhaps encountered as  a l s o r e q u i r e some e x t e r n a l  they m i g r a t e from t h e i r s i t e of o r i g i n  s i t e i n the p e r i p h e r a l t i s s u e , i n o r d e r tiation.  A l t h o u g h very  c e l l surface is  little  i s known about what p r o p e r t i e s  influence migration,  environment i s c r i t i c a l .  ability  of these c e l l s The  o f the W/W  V  gene may  be  due  the  too,  c e l l surface  I t seems r e a s o n a b l e to propose,  c h a r a c t e r i s t i c s of the  their  of  i t i s c l e a r that migration,  t h a t the o b s e r v e d e f f e c t s o f the W the s u r f a c e  to  to complete t h e i r d i f f e r e n -  a phenomenon i n which i n t e r a c t i o n between the  in  stimulation,  and  the  therefore,  to an a l t e r a t i o n  a f f e c t e d c e l l s , such t h a t  the  to respond to e x t e r n a l s t i m u l a t i o n i s a l t e r e d .  possibility  c e l l s u r f a c e has  t h a t the W l o c u s  c o n t r o l s some c h a r a c t e r i s t i c  been s u g g e s t e d b e f o r e  marrow i s n o r m a l l y r e l a t i v e l y  (Keighley  e t a l , , 1966).  u n r e s p o n s i v e even to 150x  the  con-  c e n t r a t i o n of e r y t h r o p o i e t i n t h a t i n d u c e s massive e r y t h r o p o i e s i s i n +/+  mice.  Keighley  renders W/W  V  poietin.  e_t al.  (1966) , however, r e p o r t  e r y t h r o i d stem c e l l s almost f u l l y  They suggest t h a t low  that prolonged hypoxia  responsive  oxygen t e n s i o n i t s e l f  may  to  erythro-  have some  v d i r e c t e f f e c t on  the s u r f a c e  exposing or inducing  of W/W  the f o r m a t i o n  erythroid precursors,  perhaps  of e r y t h r o p o i e t i n receptors.  Although  a number of o t h e r hypotheses can be proposed t o e x p l a i n the e f f e c t of h y p o x i a on e r y t h r o p o i e s i s , o n l y implies  the " c e l l  surface hypothesis,"  t h a t the primary a c t i o n of the W gene has  which  to do w i t h c e l l  t u r e , i s fundamental enough and b r o a d enough to account f o r the  struc-  other  v o b s e r v e d anomalies of W/W Changes i n c e l l d i s t r i b u t i o n and  mice as w e l l . surface  characteristics, particularly  degree of exposure of s u r f a c e  glycoproteins  in and  the other  133  s t r u c t u r a l m o i e t i e s , appear t o be a s s o c i a t e d w i t h the c o n t r o l o f c e l l differentiation teristics  and p r o l i f e r a t i o n .  A l t e r a t i o n s of c e l l s u r f a c e c h a r a c -  a r e c l e a r l y a s s o c i a t e d b o t h w i t h malignant  and w i t h a n t i g e n - o r mitogen-induced processes  of c e l l  N i c o l s o n , 19 75).  t r a n s f o r m a t i o n , as w e l l as w i t h  a g g r e g a t i o n and d i f f e r e n t i a t i o n  of  (Duzgunes, 19 75;  The e x p r e s s i o n o f t h e t a and o t h e r a n t i g e n s on c e l l  s u r f a c e s has been r e l a t e d t o the d i f f e r e n t i a t i o n cells  transformation  and m a t u r a t i o n  (Hammerling, 19 71; R a f f , 19 70,; James et_ a l . , 19 74), and a l o s s  some a n t i g e n s p r e s e n t d u r i n g f e t a l development i s a s s o c i a t e d w i t h  the d i f f e r e n t i a t i o n o f v a r i o u s o t h e r c e l l (capping)  of receptor s i t e s  types.  A redistribution  on lymphocytes i s a s s o c i a t e d w i t h the  i n t e r a c t i o n o f these c e l l s w i t h a n t i g e n s and mitogens. contact also i n i t i a t e s  cell proliferation a l s o appears  t h a t t h i s may be r e l a t e d t o the c o n t r o l o f  ( S c o t t e t a l . , 1973).  cells,  uncovering  the morphology and b e h a v i o u r  control of c e l l p r o l i f e r a t i o n It  Neoplastic transformation  t o cause a change i n the p r o t e i n s o f t h e plasma membranes  the t r a n s f o r m e d  altering  Cell-to-cell  a r e d i s t r i b u t i o n o f i n t r i n s i c membrane p r o t e i n s  and i t has been suggested  of  o f T-  i s easy  distinct  c h e m i c a l g r o u p i n g s , and  o f the c e l l s , and a l t e r i n g the  (Duzgunes, 19 75).  to e n v i s a g e how some a l t e r a t i o n i n the c e l l s u r f a c e  could i n t e r f e r e with  the a c t i o n o f hormones such as e r y t h r o p o i e t i n on  the c e l l , perhaps by i n t e r f e r i n g w i t h hormone b i n d i n g .  S i m i l a r l y , an  a l t e r a t i o n i n the c e l l s u r f a c e c o u l d a l t e r the i n t e r a c t i o n o f r e c e p t o r molecules w i t h a n t i g e n s and mitogens.  A defect i n migration, too,  might r e a d i l y be r e l a t e d t o some change i n t h e s u r f a c e c o n s t i t u e n t s  134  of the c e l l s , such that  the i n t e r a c t i o n of those c e l l s with surfaces  i s a l t e r e d and migration thereby  inhibited.  Relatively l i t t l e i s known, however, about the control of c e l l surface topography, or about the precise role d i f f e r e n t surface t e r i s t i c s play i n various c e l l a c t i v i t i e s . difficult  For this reason, i t i s  to speculate on p r e c i s e l y what the W gene may  not doing) to cause i t s various e f f e c t s .  charac-  be doing (or  I t i s not necessary, however,  to postulate a direct e f f e c t of the W gene on s p e c i f i c hormone receptors or on s p e c i f i c antigen and mitogen receptors.  The d i s t r i b u t i o n of  receptors over the c e l l surface, the degree to which they are exposed, and t h e i r proximity to other moieties on the c e l l surface, may a f f e c t the functioning of those receptors.  The density and  also  distri-  bution of other structures and other charged moieties over the c e l l surface may  also influence receptor function.  Any of these  charac-  t e r i s t i c s may be under the control of the W locus. Not a l l c e l l s are equally affected by the W gene.  Primordial  germ c e l l s and prospective pigment c e l l s seem to be the most profoundly affected since these c e l l s neither migrate nor p r o l i f e r a t e nor d i f f e r e n tiate.  Erythroid progenitor c e l l s are perhaps next most severely affected,  and leukocytic progenitor c e l l s next, both populations being i n t h e i r response to p r o l i f e r a t i v e stimulation.  limited  Lymphocytes themselves  are only very mildly affected, being marginally slower i n migration and somewhat a l t e r e d i n r e a c t i v i t y to antigens. respond quite normally migrate normally  Many c e l l types, however,  to t h e i r respective hormones, many appear to  or very nearly normally, and, indeed, the response  135 of T - c e l l s  to s t i m u l a t i o n appears to be  enhanced by  the W  mutation.  T h i s d i f f e r e n t i a l a c t i o n of the W l o c u s suggests t h a t the c h a r a c t e r i s t i c s which are c o n t r o l l e d by activities  o f o n l y c e r t a i n types  are c h a r a c t e r i z e d by  different  p r e c i s e combination of these than any  the W gene are of s i g n i f i c a n c e i n the of c e l l s .  D i f f e r e n t types  s u r f a c e f e a t u r e s , and  i t may  features or t h e i r i n t e r a c t i o n ,  of  cells  be  the  rather  s i n g l e s u r f a c e c h a r a c t e r , which i s most s i g n i f i c a n t  c e l l functions.  The  primary e f f e c t of the W gene, t h e r e f o r e , may  the same i n a l l c e l l s , but f a c e f e a t u r e s may  in certain be  the u l t i m a t e e f f e c t of the m u t a t i o n on  sur-  be more pronounced or more f a r - r e a c h i n g i n some c e l l s  than i n o t h e r s , at l e a s t  i n terms of i t s s i g n i f i c a n c e f o r c e r t a i n f u n c -  tions . The the  hypothesis  t h a t the W l o c u s  c e l l s u r f a c e i s the only h y p o t h e s i s  controls a characteristic y e t proposed which appears  a b l e to account f o r a l l the p l e i o t r o p i c e f f e c t s of t h i s this  reason  alone  i t i s an a t t r a c t i v e h y p o t h e s i s .  a d i f f i c u l t hypothesis  The  of W/W -type c e l l s V  in  technical  be  simply  no  u s e f u l i f s t u d i e s of the s u r f a c e  a r e t o prove f e a s i b l e .  a r e not o b t a i n a b l e  techniques  I t i s , however,  for isolating  In a d d i t i o n , c o n s i d e r a b l e  surfaces  a profound e f f e c t  and  controversy  cells  still  types  prospective  f o r i n v i t r o s t u d i e s ; there these  on  features  Other a f f e c t e d c e l l  these mice, p a r t i c u l a r l y p r i m o r d i a l germ c e l l s  pigment c e l l s ,  For  procedures.  d e m o n s t r a t i o n t h a t the W gene has  B - c e l l s , however, may  gene.  to t e s t , f o r i n v e s t i g a t i o n s of c e l l  a r e s e v e r e l y l i m i t e d by  of  from d e v e l o p i n g  e x i s t s over the  are embryos.  identity  136  of h e m a t o p o i e t i c  stem c e l l s ,  and  thropoietin-responsive cells  s p e c i f i c c e l l precursors  a r e not  readily isolated  of c e l l s  i n bone marrow.  obtained  i n l a r g e numbers from thymus, s p l e e n , and  pure B - c e l l p o p u l a t i o n s cells  are o b t a i n a b l e  a r e , t h e r e f o r e , convenient  the c e l l  cells  use  of l e c t i n s  of the more p o p u l a r  s u r f a c e s t u d i e s , and  from the v a r i e t y  on the o t h e r hand, are  to use  and  i n i n v e s t i g a t i o n s of  f e a t u r e s of c e l l  s u c c e s s f u l methods now  the use  of l e c t i n s (1975).  surfaces i s  available for  f o r t h i s purpose has Determination  cell  been  de-  of the number  and  a  of t h e i r r e d i s t r i b u t i o n f o l l o w i n g s t i m u l a t i o n , u s i n g the t e c h - •  n i q u e s d e s c r i b e d by N i c o l s o n and W/W  V  (19 75), might r e v e a l d i f f e r e n c e s between  c e l l s which c o u l d be  f o r m a t i o n which these p o p u l a t i o n s  r e l a t e d to the d i f f e r e n c e s i n t r a n s of c e l l s e x h i b i t .  The r e l a t i o n s h i p  between l e c t i n b i n d i n g , r e c e p t o r r e d i s t r i b u t i o n , and b i o l o g i c a l i s not a t a l l c l e a r :  b i n d i n g of l e c t i n s  formation.  Nevertheless,  any  d i f f e r e n c e s between +/+  t h e i r response to l e c t i n s might p r o v i d e  about the s u r f a c e f e a t u r e s of these I t has  s u r f a c e of the  and  and W/W  cells  V  some s i g n i f i c a n t  cytoplasmic  microfilaments  c e l l membrane, may  trans-  information  cells.  been s u g g e s t e d t h a t c e l l  as networks of m i c r o t u b u l e s  response  to lymphocytes, f o r example,  does not always r e s u l t i n r e c e p t o r r e d i s t r i b u t i o n or i n c e l l  in  and  These  d i s t r i b u t i o n of l e c t i n - b i n d i n g s i t e s on lymphocyte s u r f a c e s , and  +/+  readily  lymph nodes,  i n a v a r i e t y of ways.  to probe the  s c r i b e d i n d e t a i l by N i c o l s o n  study  ery-  surface. The  one  Lymphoid c e l l s ,  such as  s t r u c t u r e s , such  l o c a t e d n e a r the  c o n t r o l the m o t i l i t y  and  inner  distribution  137  of membrane p r o t e i n s  (Nicolson,  1973).  I f t h i s i s t r u e , the a c t i o n  of the W gene may be t o i n t e r f e r e w i t h the s t r u c t u r e o r f u n c t i o n o f microfilaments  or microtubules,  and t h e r e b y t o a l t e r the e f f e c t which  r e d i s t r i b u t i o n o f membrane s t r u c t u r e s n o r m a l l y e x e r t s  on the c e l l .  T h i s h y p o t h e s i s seems perhaps u n l i k e l y , s i n c e so many c e l l W/W  v  mice appear t o f u n c t i o n n o r m a l l y .  does a f f e c t c e l l c y t o p l a s m i c by  u l t r a s t r u c t u r a l studies  studies  Whether o r n o t the W l o c u s  s t r u c t u r e s , however, c o u l d be determined  of affected c e l l  In a d d i t i o n t o p r o b i n g distribution,  c e l l surface  types. receptors,  and t h e i r r e d i s t r i b u t i o n f o l l o w i n g l e c t i n  concerning  types i n  immune r e a c t i o n s  o f W/W  v  their  initial  binding,  mice might h e l p  further  i n exploring v  the c e l l s u r f a c e h y p o t h e s i s .  The a b i l i t y  respond t o B - c e l l mitogens and a n t i g e n s  of c e l l s  other  r e v e a l t h a t n o t a l l B - c e l l responses i n W/W  V  mice are d e f i c i e n t . itself  i s not  these mice, and would make the h y p o t h e s i s o f a g e n e r a l  ficiency unlikely. i n W/W  V  defect  mice t o  than LPS and SRBC might  a r e s u l t would'suggest t h a t a n t i b o d y p r o d u c t i o n in  from W/W  Such impaired  B - c e l l de-  The d e t e c t i o n o f normal responses t o some  antigens  mice would n o t be i n c o n s i s t e n t w i t h the h y p o t h e s i s t h a t t h e i n W/W  V  cells,  surface i n t e r a c t i o n .  when i t i s m a n i f e s t , l i e s  a t o r n e a r the c e l l  CONCLUSION  The of  the  p o s s i b i l i t y t h a t the  cell  s u r f a c e which i n f l u e n c e s the  environmental s t i m u l i can  W locus controls a  e x p l a i n a l l the  because i t may  i s an  exciting  known e f f e c t s  p r o v i d e a new  the If  critical  W gene does a l t e r c e l l  teration  can  be  d e f i n e d , the  and  closer  and  to b e i n g  the  the  control  cells  with  o n l y because i t  W m u t a t i o n i n mice, but  and  differentiation.  of  i f the  C e l l surface  i n many c e l l n a t u r e of  p r e c i s e r o l e of c e l l s u r f a c e and  d i f f e r e n t i a t i o n may  these processes at  understood.  138  the  also control  migratory process  differentiation  s u r f a c e s , and  t i c s i n migration, p r o l i f e r a t i o n , clarified,  of  h y p o t h e s i s , not  components of b o t h the  i n d u c t i o n of p r o l i f e r a t i o n the  interaction  system i n which to i n v e s t i g a t e the  of c e l l migration, p r o l i f e r a t i o n , phenomena are  of  characteristic  the  and types. al-  characterisbe  cellular  somewhat level  REFERENCES  A l l a n , D. , Auger, J . , and Crumpton, M. 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