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UBC Theses and Dissertations

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

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THE EFFECT OF CONTINUAL ANTIGENIC STIMULATION ON THE IMMUNE SYSTEM OF MICE by WILLIAM ROBERT McMASTER B.Sc. U n i v e r s i t y of B r i t i s h Columbia, 1973 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREEMNTS FOR THE DEGREE OF MASTER OF SCIENCE In the Department of M i c r o b i o l o g y We accept t h i s t h e s i s as conforming to the re q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA February, 1976 In p re sent ing t h i s t he s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, 1 agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree tha t permiss ion fo r ex ten s i ve copying o f t h i s t he s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r ep re sen ta t i ve s . It i s understood that copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed without my w r i t t e n permi s s ion . Department of /VA^ bio fog y / The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date Hc^/ ?X , / 7 % i i ABSTRACT The e f f e c t of c o n t i n u a l a n t i g e n i c s t i m u l a t i o n on the immune system of mice was studi e d u s i n g two d i f f e r e n t experimental approaches. A GVHR was induced i n F i mice by the i n j e c t i o n of p a r e n t a l spleen c e l l s at weekly i n t e r v a l s . Several weeks l a t e r the spleen c e l l s of mice undergoing a GVHR were shown to be immunosuppressed as t h e i r i n v i t r o responses to the mitogens Con A and LPS were s u b s t a n c i a l l y lower than c o n t r o l animals. The serum from these t r e a t e d mice was a l s o immunosuppressive to normal spleen c e l l s . The p r o l i f e r a t i v e response to Con A and a l l o g e n e i c c e l l s of normal: syngeneic, a l l o g e n e i c , and p a r e n t a l spleen c e l l s was 90% suppressed when GVH serum was added i n comparison to the a d d i t i o n of normal serum. S i m i l a r l y , the i n v i t r o antibody response to a T dependant antigen was impaired; however, the antibody response to a T independant antigen was not impaired. These r e s u l t s i n d i c a t e that T c e l l f u n c t i o n s are more s e n s i t i v e than are B c e l l f u n c t i o n to immunosuppressive f a c t o r s i n the serum of mice undergoing a GVHR. The serum was f r a c t i o n a t e d by g e l f i l t r a t i o n on a Bio-Gel P-200 column. The i n h i b i t o r y m a t e r i a l i n GVH serum e l u t e d i n the immunoglobulin f r a c t i o n of serum which i n d i c a t e that i t has a molecular weight of 150,000 or greater. The second approach studied i n v o l v e d c o n t i n u a l a l l o g e n e i c s t i m u l a t i o n . P a r e n t a l type mice were i n j e c t e d at f i v e day i n t e r v a l s w i t h F i spleen c e l l s i n order to induce a HVG r e a c t i o n . A f t e r s e v e r a l i n j e c t i o n s the spleen c e l l s from these mice were test e d i n v i t r o . The spleen c e l l s from HVG mice responded the same as normal spleen c e l l s to the mitogens Con A and LPS. The spleen c e l l s from HVG mice showed an enhanced i n v i t r o antibody response as compared to normal spleen c e l l s . This enhancement was a t t r i b u t e d to the a l l o g e n e i c e f f e c t . This s e r i e s of experiments have shown that the i n d u c t i o n of a GVHR i n mice can l a t e r l e a d to immunosuppression and production of immunosuppressive i i i f a c t o r s i n the serum of these mice. The i n d u c t i o n of a short term HVG r e a c t i o n has no adverse e f f e c t s on the immune system except f o r enhancing an antibody response. I t i s p o s s i b l e that a more prolonged HVG r e a c t i o n would p a r a l l e l the immunosuppression observed i n mice undergoing a GVHR. i v TABLE OF CONTENTS Page INTRODUCTION 1 I. The C e l l s which I n i t i a t e a GVHR ........ . . . . > . 2 I I . Fate of the Host 2 I I I . The E f f e c t of GVHR Upon the Immune Responsiveness of the Host. 4 IV. Immunosuppression i n Tumour Bearing Animals 5 V. Objective of the Thesis 6 MATERIAL AND METHODS 8 I. Animals 8 I I . I nduction of GVHD 8 I I I . Induction of Con t i n u a l A l l o g e n e i c S t i m u l a t i o n . 8 IV. C o l l e c t i o n and Pr e p a r a t i o n of Serum . . 8 V. In V i t r o Mitogen S t i m u l a t i o n 9 VI. P r e p a r a t i o n of DNP-LPS 9 V I I . In V i t r o Antibody Production 10 V I I I . Serum F r a c t i o n a t i o n 10 RESULTS I I I. Immune Responses of Animals Undergoing GVHD 11 I I . Immune Responses of Animals Undergoing Continual A l l o g n e i c S t i m u l a t i o n (HVG) 11 I I I . E f f e c t s of Serum from Normal and GVH Mice on the Immune Responses of Normal Lymphocytes 15 IV. Gel F i l t r a t i o n of Serum . . . 19 V. Assay of F r a c t i o n a t e d Serum 19 DISCUSSION 23 BIBLIOGRAPHY 26 V LIST OF TABLES Page Table I In V i t r o Immune Responses of Spleen C e l l s from HVG Mice 14 Table I I E f f e c t of Serum from Normal and GVH Mice on the In V i t r o Primary PCC Response 20 vi LIST OF FIGURES Page Figure 1. K i n e t i c s of mitogen s t i m u l a t i o n s of c o n t r o l and GVH mice 12 2. Dose response of Con A and LPS 13 3. S t i m u l a t i o n of normal spleen c e l l s by Con A i n the presence of mouse s e r u m . . . . . . . . 16 4. V i a b i l i t y of normal spleens c u l t u r e d w i t h mouse serum 17 5. I n h i b i t i o n of the MLC by GVH serum 18 6. E l u t i o n p r o f i l e of normal and GVH serum 21 7. Assay of f r a c t i o n a t e d serum 22 v i i ACKNOWLEDGEMENT I would l i k e to thank Dr. J u l i a Levy f o r her many suggestions, h e l p f u l c r i t i c i s m s and her i n v a l u a b l e encouragement during the research and w r i t i n g of t h i s t h e s i s . v i i i ABBREVIATIONS Abbre v i a t i o n s used.in t h i s t h e s i s GVHD, gr a f t - v s - h o s t disease; GVHR, gra f t - v s - h o s t r e a c t i o n ; GVH mice; mice undergoing a GVHR, GVH serum, serum from GVH mice; HCG, h o s t - v s - g r a f t ; HVG mice, mice undergoing a HVG r e a c t i o n , PHA, phytohemagglutinin, PBS, phosphate-buffered s a l i n e ; FGS, f e t a l c a l f serum; ConA, concanavalin A; LPS, b a c t e r i a l l i p o p o l y s a c c h a r i d e ; PFC, plaque forming c e l l s ; DNP-LPS, d i n i t r o p h e n y l a t e d LPS, SRBC, sheep red blood c e l l s ; MLC, mixed lymphocyte c u l t u r e ; 3H-TdR, t r i t i a t e d thymidine. 1 INTRODUCTION The i n t e n s i t y and d u r a t i o n of an immune response i s c o n t r o l l e d by s e v e r a l r e g u l a t o r y mechanisms. An immune response i s normally i n i t i a t e d when immuno-competent c e l l s are confronted w i t h an antigen. Once the antigen i s removed the immune response u s u a l l y subsides, implying that c o n t i n u a l a n t i g e n i c s t i m u l a t i o n i s necessary f o r a c o n t i n u a l immune response. C o n t r o l of t h i s l e v e l i s th e r e f o r e maintained by the source of antigen a v a i l a b l e to the lympho i d eel1s. Suppression of an antibody response by passive immunization has been observed f o r many antigens (Rowely, 1973). R e l a t i v e l y small amounts of s p e c i f i c antibody i f given w i t h i n twenty-four hours a f t e r immunization w i l l s p e c i f i c a l l y suppress the antibody response to that antigen (Rowely, 1973). This mechanism has been c a l l e d antibody feedback i n h i b i t i o n and has a l s o been shown to operate i n v i v o under normal c o n d i t i o n s (Graf, 1967). There i s a l s o evidence of a c e l l u l a r c o n t r o l system. Macrophages can modulate the immune response by processing and presenting antigen to other lymphoid c e l l s . C e r t a i n thymus-dependant lymphocytes (suppressor T c e l l s ) can d i r e c t l y e f f e c t the production of antibody by thymus independant c e l l s (B c e l l s ) (Katz, 1972). Under normal p h y s i o l o g i c a l c o n d i t i o n s an immune system i s not confronted w i t h a chronic source of antigen and th e r e f o r e a l l the c o n t r o l mechanisms act to r e g u l a t e an e f f e c t i v e immune response. One model system i n which immunocompetent c e l l s are confronted w i t h a c o n t i n u a l source of antigen i s the g r a f t verses host r e a c t i o n (GVHR). A GVHR i s induced by i n j e c t i o n of lymphoid c e l l s , which are capable of engaging i n an immune response, i n t o a host which possess antigens d i s t i n c t from the i n j e c t e d c e l l s . The host must be incapable of mounting an immune response against the grafted c e l l s i n order f o r the gra f t e d c e l l s to s u r v i v e . The simplest system to study i s to i n j e c t p a r e n t a l lymphoid c e l l s i n t o a 2 g e n e t i c a l l y t o l e r a n t F i hy b r i d host. The term GVHR r e f e r s to the immunological response of the donor lymphoid c e l l s to the f o r e i g n h i s t o c o m p a t i b i l i t y antigens of the host. The term g r a f t versus host disease (GVHD) r e f e r s to the complex syndrome which r e s u l t s i n the host as a r e s u l t of the GVHR ( E l k i n s , 1971). The C e l l s which I n i t i a t e a GVHR I t has been shown i n a number of experiments that T lymphocytes are the c e l l s r e s p o n s i b l e f o r a GVHR. Treatment of normal spleen c e l l s w i t h a n t i -t h e t a serum and complement, which k i l l s the T c e l l p o p u l a t i o n , w i l l render the c e l l s incapable of i n i t i a t i n g a GVHR (Lonai, 1973) . S i m i l a r l y lymphoid c e l l s obtained from ad u l t mice that have undergone neonatal thymecotomy are a l s o incapable of inducing a GVHR ( M i l l e r , 1967). Bone marrow c e l l s do not d i r e c t l y induce GVHR. However they c o n t a i n precursor T c e l l s which are matured by the host's thymus and then are capable of inducing a GVHR (Thomas, 1969). The theory that bone marrow c e l l s r e q u i r e the host's thymus i n order to induce a GVHR i s supported by the f a c t that bone marrow c e l l s do not induce a GVHR i n thymectomized h o s t s , ( T a y l o r , 1963). More r e c e n t l y i t has been shown that there are two types of T c e l l s which might be required to e l i c i t a GVHR. S y n e r g i s t i c responses were obtained when small numbers of c e l l s from lymphoid t i s s u e s that were r i c h i n GVH a c t i v i t y , such as spleen and lymph nodes, were combined w i t h a l a r g e r number of weekly r e a c t i v e thymus c e l l s . The degree of synergy was dependant upon the r a t i o of the two c e l l types (Cantor, 1970). Fate of the Host The i n d u c t i o n of a GVHR i n a host animal leads to a complex syndrome. Depending on the genetic d i f f e r e n c e s between the host and the donor, and the number of donor c e l l s i n j e c t e d , v a r i o u s l e s i o n s occur. With strong genetic d i f f e r e n c e s the GVHD i s c h a r a c t e r i z e d by l o s s of body weight, splemomegaly, 3 hematomegaly, lymph node atrophy, immunosuppression, g l o m e r u l o n e p h r i t i s , and ev e n t u a l l y death. Depending on the s t r a i n s df inbred mice used, v a r i o u s syndromes occur. In some s t r a i n combination, one i n j e c t i o n of donor c e l l s w i l l lead to death w i t h i n t h i r t y days whereas w i t h other s t r a i n combinations a f t e r four equivalent i n j e c t i o n s , the animals remain h e a l t h y up to one year a f t e r the l a s t i n j e c t i o n . Splenomegaly and immunosuppression are e a r l y and constant f i n d i n g s w i t h almost every animal undergoing a GVHR. I n i t i a l l y i t was assumed that the spleen enlargement r e s u l t e d from the gra f t e d c e l l s p r o l i f e r a t i n g i n response to the host a l l o a n t i g e n s (Simonsen, 1957) . I n v e s t i g a t i o n s u s i n g chromosome markers soon i n d i c a t e d that most of the p r o l i f e r a t i n g c e l l s were of host o r i g i n ( E l k i n s , 1966). More r e c e n t l y i t was shown that grafted c e l l s p r etreated w i t h mitomycin-C, which i n h i b i t s DNA sy n t h e s i s , can s t i l l induce splenomegaly i n F! hosts provided they were mixed with Fj c e l l s overnight p r i o r to i n j e c t i o n i n t o host animals ( S c o l l a y , 1974) . These authors' explanation f o r t h e i r r e s u l t s was that the i n j e c t e d c e l l s can mount an immune response to the host's a l l o a n t i g e n s (as f a r as p o s s i b l e without c e l l d i v i s i o n ) and r e l e a s e s o l u b l e f a c t o r s which non s p e c i f i c a l l y induce host c e l l p r o l i f e r a t i o n . Regardless of whether t h e i r e x p l a n ation i s t r u e , splenomagaly appears to be the r e s u l t of host c e l l p r o l i f e r a t i o n . There has been one report i n which the host appeared tomounta response to the grafted c e l l s . When p a r e n t a l lymphoid c e l l s were i n j e c t e d i n t o an F i host these c e l l s produced antibody against the host ( i f they were B c e l l s ) or become c y t o t o x i c ( i f they were T c e l l s ) . The p a r e n t a l c e l l s now have something which the F i c e l l s l a c k themselves. This i s the i d i o t y p e of the a n t i - p a r e n t a l antibody and the corresponding receptors on the parent a n t i - F i T and B lymphocytes. I t was shown that a few F i animals i n j e c t e d w i t h p a r e n t a l c e l l s produced an antibody to t h i s i d i o t y p e ( B i n z , 1975). At t h i s time i t remains unclear what r o l e i n a GVHR t h i s host response may have. 4 The E f f e c t of GVHR Upon the Immune Responsiveness of the Host The immune system of animals undergoing a GVHR has been shown to be se v e r e l y immunosuppressed. B c e l l responses, as measured by serum antibody l e v e l s and by PFC, to both T dependant antigens (Davies, 1970) and T i n -dependant antigen ( M o l l e r , 1971) have been shown to be suppressed. A l l o g r a f t r e j e c t i o n has been found to be prolonged i n animals w i t h chronic GVH, implying that T c e l l f u n c t i o n was suppressed under these c o n d i t i o n s (Lapp, 1969) . In an attempt to overcome the immunosuppression i n GVH mice, normal syngeneic lymphoid c e l l s were a d o p t i v e l y t r a n s f e r r e d i n t o the host mice (Claman, 1969) . This t r a n s f e r d i d not r e s t o r e immunocompetence of "the host im p l y i n g that the donated competent c e l l s had become immunosuppressed. In another system i t was shown that F i animals which had received one i n j e c t i o n of p a r e n t a l c e l l s were r e s i s t a n t to a second i n j e c t i o n of p a r e n t a l c e l l s which would normally cause acute GVH i n untreated animals ( F i d d , 1966). Resistance to the second challenge was t r a n s f e r r e d to normal F i animals by p a r a b i o s i s or cross c i r c u l a t i o n ( F i e l d , 1967). This experiment i n d i c a t e d that the r e s i s t a n t s t a t e was due to e i t h e r i n h i b i t o r y f a c t o r s or suppressor c e l l s i n the blood of these animals. There have been a few attempts to demonstrate i n v i v o that serum from GVH mice contains f a c t o r s which can suppress the immune system of normal animals (Grushka, 1974). These experiments were u n s u c c e s s f u l , perhaps due to i n s u f f i c i e n t amounts of serum t r a n s f e r r e d . More r e c e n t l y , i t was demonstrated that spleen c e l l s from GVH mice when c u l t u r e d i n v i t r o produce a f a c t o r ( s ) which suppresses the response of normal spleen c e l l s to PHA ( P h i l l i p s , 1975). In a d d i t i o n to the demonstration of i n h i b i t o r y f a c t o r s other workers have r e c e n t l y shown that c e r t a i n c e l l populations from the spleens of animals under-going GVHR can suppress an i n v i t r o immune response. I t was shown that the adherent c e l l s from these animals, when c u l t u r e d w i t h normal spleen c e l l s 5 would suppress the generation of PFC to the T dependant a n t i g e n , SRBC ( E l i e , 1975). The adherent c e l l s from the spleens of animals undergoing a GVHR d i d not appear to be s o l e l y suppressor c e l l s but served t o f u n c t i o n as re g u l a t o r y c e l l s i n the i n d u c t i v e phase of an immune response. This was based on t h e i r r e s u l t s which showed that these adherent c e l l s could r e p l a c e normal adherent c e l l s i f added i n c e r t a i n r a t i o s . They postulated from t h e i r i n v i t r o r e s u l t s that an excess number of the adherent c e l l s i n the host's spleen may be r e s p o n s i b l e f o r the observed i n v i v o immunosuppression of humoral responses. Summarizing these f i n d i n g s i t appears that the immune system of an animal undergoing a GVHR may be immunosuppressed due to an excess number of suppressor c e l l s which i n turn produce an i n h i b i t o r y f a c t o r ( s ) which suppresses the f u n c t i o n of other lymphoid c e l l p opulations. Immunosuppression i n Tumour Bearing Animals Another s i t u a t i o n i n which animals are subjected to excess antigen are those animals which have a p r o g r e s s i v e l y growing tumour. I t has been shown that immunosuppression o f t e n accompanies tumour growth. Tumour s p e c i f i c immunity which i s present during e a r l y growth of the tumour has been shown to decrease as the tumour s i z e increases (Deckers, 1973). In some tumour systems, along w i t h the decrease i n s p e c i f i c immunity there i s a l s o a decrease i n general immunocompetence. This general immunocompetence has been measured by la c k of a b i l i t y of spleen c e l l s from tumour bearing mice to respond non s p e c i f i c a l l y to mitogens (Whitney, 1974), to mount a response to a l l o g e n e i c c e l l s (Haran-Gheru, 1973), and to produce antibody to a defined antigen (Smith, 1973). There are s e v e r a l p o s s i b l e causes of the immunosuppression which accompanies tumour growth. Previous work has shown that serum from mice w i t h l a r g e tumours i s capable of suppressing the p r o l i f e r a t i v e response of normal mouse spleen c e l l s to mitogens and a l l o g e n e i c c e l l s (Whitney, 1975). I t has 6 been found that the m a t e r i a l r e s p o n s i b l e f o r t h i s i n h i b i t i o n e l u t e d w i t h immunoglobulin on Shephadex G-150. Furthermore, the i n h i b i t o r y f a c t o r could be removed on an anti-mouse immunoglobulin immunoadsorbent (Levy, 1975) . These r e s u l t s i n d i c a t e that the i n h i b i t o r y substance i s antibody - l i k e i n nature and could be an antibody-antigen complex or a r e g u l a t o r y antibody. The spleens, from those animals whose serum i s immunosuppressive, c o n t a i n c e l l s which can suppress the response of normal spleen c e l l s to mitogens (Pope, 1975). The r e l a t i o n s h i p between the suppressor c e l l s and the serum f a c t o r i s u n c l e a r ; however one might speculate that the serum f a c t o r was a product of the suppressor c e l l s i n response to excess antigen. Another source of the serum f a c t o r which cannot be ignored i s that i t could be a product from the tumour c e l l s . This i s u n l i k e l y , s i n c e these suppressive substances can be removed on immunoadsorbent columns prepared w i t h anti-mouse immunoglobulin. Objective of the Thesis The o b j e c t i v e of t h i s t h e s i s was to study the e f f e c t s on the immune system of c o n t i n u a l a n t i g e n i c s t i m u l a t i o n s . Two model systems were used: (1) A GVHR was induced i n F i animals by the i n j e c t i o n of p a r e n t a l spleen c e l l s . This enabled the grafted c e l l s to be i n constant contact w i t h a l l o g e n e i c antigen w h i l e the host was g e n e t i c a l l y t o l e r a n t t o the grafted c e l l s . (2) Animals (mice) were subjected to c o n t i n u a l a l l o g e n e i c s t i m u l a t i o n by repeated a d m i n i s t r a t i o n of semi-allogeneic ( F i ) spleen c e l l s . This i n i t i a t e d a one way "Host vs. G r a f t " response since the i n j e c t e d c e l l s were t o l e r a n t ' t o the host. The lymphoid c e l l s of these animals were then assayed by v a r i o u s i n v i t r o measurements of immunocompetance. I t w i l l be shown that c o n t i n u a l a l l o g e n e i c s t i m u l a t i o n s has no adverse e f f e c t s on the immune system of mice w h i l e the 7 i n d u c t i o n of a GVHR i n mice leads to immunosuppression. 8 MATERIALS AND METHODS Animals BALB/c, DBA/2J, CBA/J, C57BL/6J, and C57BL/6 X DBA/2J F i (B6D2 ¥ x) Female mice (Jackson Laboratory, Bar Harbour, Maine) aged 2-4 months were used throughout t h i s study. Induction of GVHD GVHD was induced i n F i animals by four weekly i n t r a p e r i t o n e a l i n j e c t i o n s of 50 x 10 6 p a r e n t a l (DBA/2J) spleen c e l l s . These mice w i l l be r e f e r r e d to as GVH mice. C o n t r o l mice c o n s i s t e d of F i animals i n j e c t e d as above w i t h 50 x 10 6 Fx spleen c e l l s . Mice undergoing GVHD induced i n t h i s way were c l i n i c a l l y w e l l , although spleen s i z e at time of s a c r i f i c e was moderately enlarged (2-3 fold' increase i n weight over normal c o n t r o l s ) . Induction of Con t i n u a l A l l o g e n e i c S t i m u l a t i o n C o n t i n u a l a l l o g e n e i c s t i m u l a t i o n was induced i n DBA/2J mice by 7 i n j e c t i o n s , at 5 day i n t e r v a l s of 20 x 10 6 F i spleen c e l l s . These mice w i l l be r e f e r r e d t o as HVG mice. C o l l e c t i o n and Pre p a r a t i o n of Serum Blood was c o l l e c t e d from the r e t r o - o r b i t a l sinus of normal, c o n t r o l , and GVH mice 4 and 6 weeks a f t e r the l a s t i n j e c t i o n of c e l l s . The blood was allowed to c l o t f o r 2 hours at 4?C a f t e r which the serum was separated and pooled i n t o the three groups. I t was heat i n a c t i v a t e d at 56°C f o r 30 min, absorbed w i t h SRBC f o r 30 min and d i a l y z e d against RPMI 1640 media f o r 24 hours. Fo l l o w i n g d i a l y s i s the serum was u l t r a c e n t r i f u g e d at 100,000 x g f o r 3 hours, f i l t e r e d through a m i l l i p o r e 0.22 u f i l t e r and stored at -20°C. 9 In V i t r o Mitogen S t i m u l a t i o n s Spleen c e l l s were prepared i n PBS + 5% FCS. The c e l l s were c e n t r i f u g e d at 2000 x g f o r 5 min, resuspended i n PBS + 5% FCS, and the v i a b i l i t y determined by trypan blue e x c l u s i o n . Desired number of c e l l s were c e n t r i f u g e d and resus-pended i n RPMI 1640 medium and dispensed i n t o m i c r o t i t e r p l a t e s (Linbro Chemical Co.). Each w e l l contained 5 x 10 5 v i a b l e spleen c e l l s i n a f i n a l volume of 0.25 ml medium w i t h FCS at a f i n a l c o n c e n t r a t i o n of 2%, and appropriate concentrations of Con A, LPS, or mouse serum. The c u l t u r e s were incubated at 37°C i n a hu m i d i f i e d atmosphere of 95% a i r - 5% CO2 f o r 1-4 days as i n d i c a t e d i n each experiment. Eighteen hours before the c e l l s were harvested 1.0 u C i of 3H-Thymidine (sp. a c t . 5.0 Ci/mmol, New England Nuclear, Montreal, Canada) was added to each w e l l . The c e l l s were harvested onto glass f i b e r f i l t e r paper, d r i e d , and the r a d i o a c t i v i t y determined. Two way MLC were performed as described above by c u l t u r i n g together 2.5 x 10 s CBA/J spleen c e l l s and 2.5 x 10 5 BALB/c spleen c e l l s and h a r v e s t i n g a f t e r 4 days i n c u b a t i o n . Tests were run i n t r i p l i c a t e , and the r e s u l t s expressed as the mean value. The standard e r r o r i n a l l instances was always < 10%. Preparation of DNP-LPS LPS, E. c o l i 055:B5, (Difco L a b r a t o r i e s ) was d i s s o l v e d to a concentration of 20 mg/ml i n b o r a t e - s a l i n e (pH 8.5). 2 , 4 - d i n i t r o b e n z e n e - s u l f o n i c a c i d (Eastman Kodak Co.) and sodium carbonate ( F i s h e r Chemicals) were then each added to a f i n a l c o n c e n t r a t i o n of 20 mg/ml. The r e a c t i o n was s t i r r e d f o r 24 hours at room temperature and unreacted reagents were removed by exhaustive d i a l y s i s a g ainst b o r a t e - s a l i n e f o r 48 hours. The DNP-LPS was then s t e r i l i z e d by m i l l i p o r e f i l t r a t i o n and stored at -20°C. I t has been shown that DNP-LPS cu l t u r e d w i t h normal spleen c e l l s at sub mitogenic doses invokes a DNP s p e c i f i c antibody response without the help of T lymphocytes (T independant response); (Jacobs, 1975) . 10 In V i t r o Antibody Production Spleen c e l l s were prepared i n RPMI 1640 + 5% FCS buffered w i t h 35 mM Hepes b u f f e r . C e l l s were c u l t u r e d at a conc e n t r a t i o n of 10 x 10 6 i n 35 mm p e t r i p l a t e s (Falcon #3301) c o n t a i n i n g 2.0 ml RPMI 1640 media supplemented w i t h 10% FCS ( M i c r o b i o l o g i c a l A s s o c i a t e s #84557), 5 x 10~ 5 M 2-mercaptoethanol, and v a r y i n g percentages of mouse serum. Antigens used were e i t h e r 5 x 10 6 SRBC or 0.10 Ug/ml DNP-LPS. Cu l t u r e s were incubated at 37°C on a r o c k i n g p l a t f o r m f o r 4 days. The number of d i r e c t PFC was determined w i t h a microscope s l i d e assay (Cunninghamm, 1968). DNP plaques were determined using SRBC coated w i t h d i n i t r o p h e n y l a t e d r a b b i t anti-SRBC-Fab 1 (Strausbach, 1970). S p e c i f i c anti-DNP plaques were enumerated by s u b t r a c t i n g background SRBC plaques from the t o t a l number obtained. Serum F r a c t i o n a t i o n Samples of both normal F i and GVH serum were chromatographed on an acending Bio-Gel P-200 column (2.6 cm x 90 cm) usin g b o r a t e - s a l i n e (pH 8.5) as the e l u t i n g b u f f e r . The sample volumes were 1 ml, e l u t i o n flow r a t e was 10 ml/hour and 2.0 ml f r a c t i o n s c o l l e c t e d . Peaks of 280 nm absorbing m a t e r i a l e l u t i n g from the column were pooled and concentrated by p r e c i p i t a t i o n w i t h 50% saturated ammonium s u l f a t e . The p r e c i p i t a t e s were kept at 4°C f o r 18 hours then c e n t r i f u g e d at 18,000 x g f o r 20 min. The p e l l e t s were d i s s o l v e d i n b o r a t e - s a l i n e and d i a l y z e d against the same. The d i a l y z e d m a t e r i a l s were made up to the o r i g i n a l volumes of the serum sample, f i l t e r e d s t e r i l i z e d , and stored at -20°C. To assay f o r i n h i b i t o r y a c t i o n , . t h e appropriate f r a c t i o n s were d i l u t e d i n 1640 medium and added to spleen c e l l c u l t u r e s e x a c t l y as described above. 11 RESULTS Immune Responses of Animals Undergoing GVHD The spleen c e l l s of c o n t r o l and GVH mice, obtained 8 weeks a f t e r the l a s t i n j e c t i o n of c e l l s , were stimulated i n v i t r o w i t h the T c e l l mitogen Con A, and the B c e l l mitogen LPS. Both c o n t r o l and GVH c e l l s f o l l o w s i m i l a r k i n e t i c s shown i n F i g . 1.as the maximal 3H-Thymidine i n c o r p o r a t i o n occurred on day 3 f o r each mitogen and each c e l l type. The maximum Con A response of c e l l s from GVH mice was 40% of the maximum response of c e l l s from c o n t r o l mice. The LPS response of GVH c e l l s was somewhat l e s s s e v e r e l y suppressed, being 73% of the response of c o n t r o l c e l l s . The suppression of GVH c e l l s i s not due to a d i f f e r e n c e i n dose response. C e l l s from c o n t r o l and GVH mice respond o p t i m a l l y to the same conc e n t r a t i o n of Con A and to the same concentrations of LPS ( F i g . 2 ) . P r e l i m i n a r y data showed that these dose responses are the same_ regardless of the day on which the c e l l s were harvested. Immune Responses of Animals Undergoing Continual A l l o g e n e i c S t i m u l a t i o n (HVG) The spleen c e l l s of normal and HVG mice, obtained 4 days a f t e r the l a s t i n j e c t i o n of F i c e l l s , were sti m u l a t e d i n v i t r o w i t h Con A and LPS. These r e s u l t s are shown i n Table I . C e l l s from the HVG animals showed a higher response to both Con A and LPS and a l s o had a higher spontaneous 3H-Thymidine uptake. Spleen c e l l s from normal and HVG mice were a l s o t e s t e d f o r an i n v i t r o antibody response to SRBC. These r e s u l t s are a l s o shown i n Table I . The c e l l s from HVG mice showed a much higher antibody response, being almost twice as high as the response of normal c e l l s . A 0 . 1 2 3 DAYS FIGURE 1. K i n e t i c s of mitogen stimulations of spleen c e l l s from co n t r o l mice (data from 4 mice), • - — • ; and of spleen c e l l s , from GVH mice (data from 9 mice), 1 A. (A) c e l l s cultured with 20 Ug/ml LPS. (B) c e l l s cultured with 2.0 Ug/ml Con A. 13 o i O 2 CL U z o K-< oc O a. oc O u z z Q > x r-I X n 1 2 4 CON A (ug/ml) FIGURE 2. Dose response of mitogen s t i m u l a t i o n s of spleen c e l l s from c o n t r o l mice (data from 4 mice), and of spleen c e l l s from GVH mice (data from 9 mice), A i . (A) LPS dose response (B) Con A dose response. IN VITRO IMMUNE RESPONSES OF SPLEEN CELLS FROM HVG MICE Spleen C e l l s From Mitogen Responses 3H-TdR Uptake (cpm) Antibody Responses P f c / c u l t u r e Normal Mice HVG Mice Medium LPS Con A Medium SRBC 8,720 53,177 82,093. 17,789 61,594 125,245 226 4,146 526 8,737 Table I The i n v i t r o mitogen and antibody responses of spleen c e l l s from HVG mice. 13 E f f e c t s of Serum from Normal and GVH Mice of the Immune Responses of Normal  Lymphocytes The f o l l o w i n g experiments were designed to t e s t whether GVH serum i s immunosuppressive to lymphoid c e l l s from normal mice. Serum obtained from normal, c o n t r o l , or GVH mice was c u l t u r e d w i t h normal F i , or DBA/2J spleen c e l l s and v a r i o u s amounts of Con A. The r e s u l t s of these experiments are shown i n F i g . 3. The optimal response to Con A of normal F i spleen c e l l s was suppressed by the a d d i t i o n of GVH serum as compared to the a d d i t i o n s of normal serum. The response of c e l l s c u l t u r e d w i t h 2% GVH serum was 30% of the response of c e l l s c u l t u r e d w i t h 2% normal serum. Inc r e a s i n g the con c e n t r a t i o n of GVH serum to 4% was even more immunosuppressive as the response was 9% of the response of c e l l s c u l t u r e d w i t h 4% normal serum. GVH serum a l s o i n h i b i t e d the Con A response of normal DBA/2J spleen c e l l s which are the same genotype as those c e l l s used to induce the GVH. The optimal Con A response of these c e l l s c u l t u r e d w i t h 4% GVH serum was 60% of the response of c e l l s c u l t u r e d w i t h 4% normal serum. In these and other experiments c u l t u r e s c o n t a i n i n g normal serum gave the same r e s u l t s as c u l t u r e s c o n t a i n i n g c o n t r o l serum, there f o r e the c o n t r o l i n a l l future experiments i s termed normal serum. Neither of the mouse sera were c y t o t o x i c to normal F i spleen c e l l s , as the v i a b i l i t i e s of c e l l s c u l t u r e d w i t h normal or GVH serum p a r a l l e l e d the v i a b i l i t i e s of c e l l s c u l t u r e d alone w i t h FCS ( F i g . 4 ) . GVH serum was a l s o t e s t e d f o r i t s a b i l i t y to i n h i b i t the mixed lymphocyte r e a c t i o n between a l l o g e n e i c c e l l s . These r e s u l t s are shown i n F i g . 5 and are expressed as a percentage response of c u l t u r e s c o n t a i n i n g normal mouse serum. As i s evident, at 4% GVH serum co n c e n t r a t i o n the response i s 93% suppressed. Further experiments were c a r r i e d out to determine whether GVH serum i n h i b i t e d T c e l l f u n c t i o n and/or B c e l l f u n c t i o n , by assaying f o r the i n 2 4 8 16 32 64 CON A (ug/ml) FIGURE 3. Stimulation of normal spleen cells by various amounts of Con A in the presense of serum from normal mice, •—-•; or in the presence of serum from GVH mice, L A. (A) normal B6D2 Fi spleen cells cultured with 2% mouse serum. (B) normal B6D2 Fi spleen cells cultured with 4% mouse serum. (C) normal DBA/2J spleen cells cultured with 4% mouse serum. 0 1 2 3 DAYS 4 FIGURE 4. V i a b i l i t y of normal B6D2 F i spleen c e l l s c u l t u r e d w i t h 0% mouse serum, H B; c u l t u r e d w i t h 4% serum from normal mice, • •; and c u l t u r e d w i t h 4% serum from GVH mice, A A. 18 0 .25 .5 1 2 4 SERUM CONCENTRATION % FIGURE 5. I n h i b i t i o n by serum from GVH mice of the mixed lymphocyte r e a c t i o n between Balb/c and BCA/J spleen c e l l s . The r e s u l t s are expressed as % of the response of spleen c e l l s c u l t u r e d w i t h corresponding amounts of serum from normal mice. 19 v i t r o generation of PFC i n i t s presence. Spleen c e l l s from normal F i mice were c u l t u r e d w i t h v a r i o u s percentages of normal or GVH serum.and stimulated w i t h e i t h e r the T dependent a n t i g e n , SRBC, or the T independent a n t i g e n , DNP-LPS. These r e s u l t s are shown i n Table 2. Although both normal and GVH serum i n h i b i t the response to SRBC, at equal concentrations the GVH serum i s always much more suppressive. The response to DNP-LPS was not suppressed by any concentration of GVH serum assayed but showed a somewhat depressed response w i t h 4% normal serum. Gel F i l t r a t i o n of Serum Figure 6 shows the e l u t i o n p r o f i l e of normal and GVH serum el u t e d from a Bio-Gel P-200 column. The f i r s t peak corresponds to the immunoglobulin f r a c t i o n of serum which contains molecules having a molecular weight of approximately 150,000 or greater. The second peak corresponds to the albumin f r a c t i o n of serum which contains molecules having a molecular weight smaller than 100,000. The f i r s t peak of GVH serum appears to have more m a t e r i a l than does peak 1 of normal serum while the second peaks appear to equal amounts of m a t e r i a l . Assay of F r a c t i o n a t e d Serum The P-200 f r a c t i o n s of normal and GVH serum were test e d f o r t h e i r a b i l i t y to i n h i b i t the p r o l i f e r a t i v e response of normal F i spleen c e l l s to Con A. These r e s u l t s are shown i n Figure 7. At higher c o n c e n t r a t i o n s , f r a c t i o n I from GVH serum, was immunosuppressive, i n h i b i t i n g the Con A response by 40% as compared to f r a c t i o n I from normal serum. By expressing the r e s u l t s as a percent of response obtained w i t h normal f r a c t i o n s i t appears that f r a c t i o n I I from GVH serum i s s t i m u l a t o r y . However the t o t a l uptake of H-thymidine was always higher i n c o n t r o l c u l t u r e s which d i d not cont a i n any mouse serum f r a c t i o n s . F r a c t i o n I I from GVH serum i s the r e f o r e not as suppressive as f r a c t i o n I I from normal serum. 20 EFFECT OF SERUM FROM NORMAL AND GVH MICE ON THE IN VITRO PRIMARY PFC RESPONSE SRBC PFC/CULTURE DNP PFC/CULTURE % Serum Added Normal GVH Normal GVH 0.5 4350 + 665 2750 269 5544 + 1053 5048 + 155 1.0 4166 + 604 1278 ± 111 5925 + 420 6743 + 283 2.0 2641 + 634 471 ± 40 6049 + 241 6898 + 641 4.0 695 + 69 76 ± 11 3717 + 1115 6357 + 340 Table I I The Influence of serum from normal and GVH mice on the development of PFC i n v i t r o to SRBC and DNP-LPS. The response i n unstimulated c u l t u r e s was always < 10% of the corresponding stimulated c u l t u r e . 21 1.5 NORMAL SERUM 1.0 0.5 < H 1.5 1.0 0.5 10 20 30 kO. 50 60 70 -FRACTION NUMBER FIGURE 6. -E l u t i o n p r o f i l e of normal and GVH serum from a Bio-Gel P-200 column. 22 21 4% 82 7 FINAL C O N C E N T R A T I O N fO FIGURE 7. E f f e c t of f r a c t i o n I and I I from Bio-Gel P-200 on the Con A response of normal F i spleen c e l l s . The r e s u l t s are expressed as % of the response obtained w i t h an equivalent f r a c t i o n of normal serum i n c u l t u r e w i t h normal 1 vmphoc 23 DISCUSSION The r e s u l t s shown i n F i g . 1 and 2 demonstrate that the spleen c e l l s from GVH mice are impaired i n t h e i r a b i l i t y to p r o l i f e r a t e i n v i t r o i n response to mitogens. T c e l l s may be more a f f e c t e d than B c e l l s because the Con A responses are more suppressed than the LPS responses. These r e s u l t s c o r r e l a t e w e l l w i t h numerous i n v i v o s t u d i e s which show that both T c e l l (Lapp, 1969) and B c e l l (Davies, 1970 and M o l l e r , 1971) responses are suppressed i n GVH mice. Recent s t u d i e s on murine leukemia v i r u s (MuLV), which i s a c t i v a t e d i n some mice by ch r o n i c GVH disease, a l s o showed that the spleen c e l l s of these mice are immunosuppressed ( P h i l l i p s , 1975). I t has been reported that i n the mouse s t r a i n combinations used i n the present study, l e s s than 40% of GVH mice had detectable l e v e l s of MuLV ( P h i l l i p s , 1975). I t i s u n l i k e l y that immuno-suppression observed i n GVH mice i s due to MuLV because one would expect, w i t h the s t r a i n combinations used i n t h i s study, l e s s than 40% of the mice to be immunosuppressed, whereas i t was found that every GVH mouse was immunosuppressed. The spleen c e l l s from mice i n j e c t e d w i t h a l l o g e n e i c F i c e l l s show a normal or increased i n v i t r o response to mitogens as compared to untreated mice. The increase i n 3H-Thymidine uptake i n unstimulated c u l t u r e s of i n j e c t e d mice i s probably the r e s u l t of an ongoing immune response to the.-allogeneic c e l l s . The increased mitogen responses may be a r e f l e c t i o n that there i s a more a c t i v e p opulation of T and B c e l l s i n the spleens of these animals. The increased number of PFC i n c u l t u r e s from i n j e c t e d mice can be a t t r i b u t e d to the " a l l o g e n e i c e f f e c t . " This enhancement of i n v i t r o antibody responses i s the r e s u l t of n o n - s p e c i f i c enhancing f a c t o r s secreted by a l l o -a c t i v a t e d T c e l l s (Katz, 1972). I t becomes apparent that a c o n t i n u a l a l l o g e n e i c s t i m u l a t i o n has no d e l e t e r i o u s e f f e c t s on the immune system of such t r e a t e d mice. A GVHR does 24 however have some e f f e c t on the immune system of these i n j e c t e d mice. In t h i s s i t u a t i o n the mice become immunosuppressed, however t h i s may be b e n e f i c i a l i n that the gra f t e d c e l l s a l s o become immunosuppressed and th e r e f o r e are no longer capable of r e a c t i n g against the host. The mice undergoing a GVHR were not immunosuppressed u n t i l s e v e r a l weeks a f t e r the i n i t i a t i o n of the GVHR. Perhaps i f the a l l o g e n e i c s t i m u l a t i o n was c a r r i e d out f o r t h i s same length of time, immunosuppressions would have a l s o occurred. The serum, from animals whose spleen c e l l s are immunosuppressed due to GVHD, i s capable of i n h i b i t i n g the f u n c t i o n of normal syngeneic, semi-syngeneic, and a l l o g e n e i c spleen c e l l s . The suppressive a b i l i t y of t h i s serum cannot be d i r e c t l y a t t r i b u t e d to i n f e c t i o u s v i r u s because the serum was u l t r a -c e n t r i f u g e d before i t was assayed. S i m i l a r l y , low molecular weight t o x i c by products not removed by the kidneys which are sometimes damaged by chronic GVH (Lewis, 1968) and s t e r o i d s would have been removed d u r i n g the d i a l y s i s against t i s s u e c u l t u r e medium. At the concentrations assayed, GVH serum appears to a f f e c t T c e l l f u n c t i o n r a t h e r than B c e l l f u n c t i o n i n that i t i n h i b i t s the p r o l i f e r a t i v e response of normal spleen c e l l s to Con A, i n h i b i t s the mixed lymphocyte r e a c t i o n , and i n h i b i t s the antibody response to a T dependent antigen but not to a T independent antigen. At higher serum concentrations B c e l l f u n c t i o n might become suppressed i n d i c a t i n g T c e l l s are more s e n s i t i v e s to the serum f a c t o r or e l s e there may be another f a c t o r a f f e c t i n g B c e l l s . I t cannot be ru l e d out at t h i s time that the i n h i b i t o r y m a t e r i a l could be a f f e c t i n g macro-phages which are sometimes necessary f o r the generation of antibody responses. However, the a d d i t i o n of 2 mercaptoethanol, which was included i n the c u l t u r e s , has been shown to e l i m i n a t e the need f o r macrophages i n v i t r o (Chen, 1972) . The i n h i b i t o r y e f f e c t s of GVH serum i s not H-2 s p e c i f i c because i t caused a 93% i n h i b i t i o n of the 2 way MLC i n which one of the responding c e l l s 25 was a l l o g e n e i c to the source of serum. Only a 50% i n h i b i t i o n of the MLC would have occurred i f the serum i n h i b i t o r was H-2 s p e c i f i c . The serum f a c t o r r e s p o n s i b l e f o r the immunosuppression does not appear to be a l l o a n t i b o d y , which i s sometimes present i n the serum of GVH mice (Lapp, W.S., Personal Communication), because the GVH serum i n h i b i t e d the Con A response of normal DBA/2J c e l l s , which are of the same genotype as the c e l l s used to induce the GVH. The i n h i b i t o r y m a t e r i a l i n the serum of GVH mice e l u t e d on a Bio-Gel P-200 column w i t h the immunoglobulin f r a c t i o n of serum. This i n d i c a t e s that the i n h i b i t o r y m a t e r i a l has a molecular weight of 150,000 or greater. Although immunoglobulin i s the main component of t h i s f r a c t i o n there are s e v e r a l other p r o t e i n s w i t h s i m i l a r molecular weights such as the complement p r o t e i n s . Further p u r i f i c a t i o n of the i n h i b i t o r y m a t e r i a l i s needed before one can conclude any a d d i t i o n a l i n f o r m a t i o n o n . i t ' s chemical nature and source. I t i s plausable to assume that i n h i b i t o r y m a t e r i a l i n the serum of GVH mice i s r e s p o n s i b l e f o r the generali z e d immunosuppression which accompanies GVHD. A s i m i l a r f a c t o r ( s ) , obtained from c e l l f r e e supernants of c u l t u r e d spleen c e l l s from GVH mice, has been r e c e n t l y reported which suppresses the PHA response of normal spleen c e l l s ( P h i l l i p s , 1975). This f a c t o r ( s ) might be an immunoregulatory molecule produced by suppressor c e l l s or i t may a c t i v a t e suppressor c e l l s which i n t u r n suppress the f u n c t i o n of other lymphoid c e l l s . I t has a l s o been found that the serum of tumour bearing animals a l s o contains a f a c t o r which s i m i l a r l y e l u t e s w i t h immunoglobulin and suppresses the f u n c t i o n of normal lymphoid c e l l s (Levy, 1975) . These immunoregulating molecules may be produced by a host i n response to an abnormal a n t i g e n i c load such as tumour or i n the case of an animal undergoing a GVHR be produced by the g r a f t e d c e l l s which are i n constant contact w i t h f o r e i g n antigen. 26 BIBLIOGRAPHY 1. B i n z , H., W i g z e l l , H. 1975. Shared i d i o t y p i c determinants on B and T lymphocytes r e a c t i v e against the same a n t i g e n i c determinants. J . Exp. Med. 142:197. 2. Cantor, H., Asofsky, R. 1970. Synergy among lymphoid c e l l s mediating the GVH response. J . Exp. Med. 131 :215. 3. Chen, C., H i r s c h , J.G. 1972. The e f f e c t s of mercaptoethanol and of p e r i t o n e a l macrophages on the antibody-forming c a p a c i t y of non-adherent mouse spleen c e l l s i n v i t r o . J . Exp. Med. 136:604. 4. Claman, H.N., Chaperon, E.A., Hayes, L.L. 1969. Thymus-marrow immuno-competence. IV. The growth and immunocompetence of t r a n s f e r r e d marrow, thymus, and spleen c e l l s i n parent and F i h y b r i d mice. T r a n s p l a n t a t i o n 7_:87. 5. Cunningham, A.J., Szenberg, A. 1968. Further improvements i n the plaque technique f o r d e t e c t i n g s i n g l e antibody-forming c e l l s . Immunology. 14/. 599. 6. Davis, W.E., Cole, L . J . , S c h a f f e r , W.T. 1970. GVHR i n n o n - i r r a d i a t e d mice. E a r l y suppression of Jerne plaques and hemopoietic colony-forming u n i t s . T r a n s p l a n t a t i o n . 9_:529. 7. Deckers, J . , Davies, R.C., Parker, G.A. 1973. The e f f e c t of tumour s i z e on concommitant tumour immunity. Cancer Res. 33 :33. 8. E l i e , R., Lapp, W.S. 1975. GVH immunosuppression: r e g u l a t o r y f u n c t i o n of macrophages i n the humoral immune response i n Immune Rec o g n i t i o n, Academic Press, Inc. 9. E l k i n s , W.L. 1966. I n t e r a c t i o n of donor and host lymphoid c e l l s i n the pathogenesis of r e n a l c o r t i c a l d e s t r u c t i o n induced by a l o c a l GVHR. J. Exp. Med. 123:103. 10. E l k i n s , W.L. 1971. C e l l u l a r immunology and the pathogenesis of GVHR. Prog. A l l e r g y . 15:81. 11. F i e l d , E.O., G i w s , J.E. 1966. Reduced s e n s i t i v i t y of F i h y b r i d r a t s to re-challenge w i t h p a r e n t a l s t r a i n spleen c e l l s . C l i n . Exp. Immunol. 1_:195. 12. F i e l d , E.O., L a u c h i , M.N., Gibbs, J.E. 1967. The t r a n s f e r of r e f r a c t o r i n e s s to GVHD i n 7i h y b r i d r a t s . T r a n s p l a n t a t i o n . 5_:241. 13. Graf, M.W., Uhr, J.W. 1969. Regulation of antibody formation by serum antibody. J . Exp. Med. 130:1175. 14. Frushka, M., Lapp, W.S. 1974. E f f e c t of lymphoid t i s s u e t r a n s p l a n t s on the i n t e n s i t y of an e x i s t i n g GVHR. T r a n s p l a n t a t i o n . 17 :157 . 15. Haran-Ghera, N. , Ben-Yaakov, A., Pele d , A. , Bentwick, Z. 1973. Immune st a t u s of SJL/J mice i n r e l a t i o n t o age and spontaneous tumour development. J . N a t l . Cancer I n s t . 50:1227. 27 16. Jacobs, D.M., Mor r i s o n , D.C.J. 1975. S t i m u l a t i o n of a T-independent primary anti-hapten response i n v i t r o by TNP-lipopolysaccharide (TNP-LPS). J . Immunology. 114:360. 17. Katz, D.H., Bencerraf, B. The r e g u l a t o r y i n f l u e n c e of a c t i v a t e d T c e l l s on B c e l l responses to antigen. Adv. Immunol. 15:1. 18. Lapp, W.S., M o l l e r , G. 1969. Prolonged s u r v i v a l of H-2 incompatible s k i n g r a f t s of F i animals t r e a t e d w i t h p a r e n t a l lymphoid c e l l s . Immunology. 17_: 339 . 19. Lewis, R.M., Armstrong, M.V.K., Andre-Schwartz, J . , Muftvofhv, A., B e l d o t t i , L., Schwartz, R.S. 1968. Chronic a l l o g e n i c d isease. I. Development of g l o m e r u l o n e p h r i t i s . J . Exp. Med. 128 :653. 20. Levy, J.G., Smith, A.G., Whitney, R.B., McMaster, R., K i l b u r n , D.G. 1976. C h a r a c t e r i z a t i o n of a T-lymphocyte i n h i b i t o r i n the serum of tumour bearing mice. Immunology, i n press. 21. L o n a i , P., Mogilner, B., R o t t e r , V., T r a i n i n , H. 1973. Studies on the e f f e c t of a thymic f a c t o r on d i f f e r e n t i a t i o n of thymus-derived lymphocytes. Eur. J . Immunol. 3_:21. 22. M i l l e r , J.A.F.P., M i t c h e l l , G.F., Weiss, G.P. 1967. C e l l u l a r b a s i s of the immunological d e f e c t s i n thymectomized mice. Nature. 214 :992. 23. M o l l e r , G. 1971. Suppressive e f f e c t of GVHR on the immune response to heterologous red c e l l s . Immunology. 20 :597 . 24. P h i l l i p s , S.M., Gleichmann, H., H i r s c h , M.S., Block, P., M e r r i l , J.P., Schwartz, R.S., Carpenter, C B . 1975. C e l l u l a r immunity i n the mouse. V. Further s t u d i e s on leukemia v i r u s a c t i v a t i o n i n a l l o g e n e i c r e a c t i o n s of mice s t i m u l a t o r y parameters. C e l l . Immunol. 15 :169. 25. Pope, B.L., Whitney, R.B., Levy, J.G. 1976. Suppressor c e l l s i n the spleens of tumour bearing mice. J . Immunology, i n press. 26. Rowely, D.A., F i t c h , F.W., S t u a r t , F.P., Kohler, H., Coseyza, H. 1973. S p e c i f i c suppression of immune responses. Science. 181:1133. 27. S c o l l a y , R.G., Hoffman, F., Globerson, A. 1974. GVHR i n F i r e c i p i e n t s i n the absence of donor (parental) c e l l p r o l i f e r a t i o n . Eur. J . Immunol. 4_:490. 28. Simonsen, M. 1957. GVHR, t h e i r n a t u r a l h i s t o r y and a p p l i c a b i l i t y as t o o l s i n research. Acta. P a t h o l . M i c r o b i o l . Scand. 40:480. 29. Smith, R.T., Kondu, S.- 1973. The s t i m u l a t o r y e f f e c t s of bearing primary methylcholanthrene-induced tumours upon the murine l y m p h o r e t i c u l a r system. I n t . J . Cancer. 12:577. 30. Strausbach, P.A., S u l i c a , A., G i v a l , D. 1970. General method f o r the de t e c t i o n of c e l l s producing antibody against haptens and p r o t e i n s . Nature. 227:68. 31. T a y l o r , R.B. 1963. Immunological competence of thymus c e l l s a f t e r t r a n s f e r to thymetomized animals. Nature. 199:873. 28 32. Thomas, E.D., Storb, R. , E p s t e i n , R.B. 1969. Symposium on bone marrow t r a n s p l a n t a t i o n . Transplant. Proc. 1_:31. 33. Whitney, R.B., Levy, J.G., Smith, A.G. 1974. Influence of tumour s i z e and s u r g i c a l r e s e c t i o n on cell-mediated .immunity i n mice. J . N a t l . Cancer I n s t . 53:111. 34. Whitney, R.B., Levy, J.G. 1975. E f f e c t s of sera from tumour be a r i n g mice on mitogen and a l l o g e n e i c c e l l s t i m u l a t i o n of normal lymphoid c e l l s . J . N a t l . Cancer I n s t . 54:733. 

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