{"Affiliation":[{"label":"Affiliation","value":"Science, Faculty of","attrs":{"lang":"en","ns":"http:\/\/vivoweb.org\/ontology\/core#departmentOrSchool","classmap":"vivo:EducationalProcess","property":"vivo:departmentOrSchool"},"iri":"http:\/\/vivoweb.org\/ontology\/core#departmentOrSchool","explain":"VIVO-ISF Ontology V1.6 Property; The department or school name within institution; Not intended to be an institution name."},{"label":"Affiliation","value":"Microbiology and Immunology, Department of","attrs":{"lang":"en","ns":"http:\/\/vivoweb.org\/ontology\/core#departmentOrSchool","classmap":"vivo:EducationalProcess","property":"vivo:departmentOrSchool"},"iri":"http:\/\/vivoweb.org\/ontology\/core#departmentOrSchool","explain":"VIVO-ISF Ontology V1.6 Property; The department or school name within institution; Not intended to be an institution name."}],"AggregatedSourceRepository":[{"label":"Aggregated Source Repository","value":"DSpace","attrs":{"lang":"en","ns":"http:\/\/www.europeana.eu\/schemas\/edm\/dataProvider","classmap":"ore:Aggregation","property":"edm:dataProvider"},"iri":"http:\/\/www.europeana.eu\/schemas\/edm\/dataProvider","explain":"A Europeana Data Model Property; The name or identifier of the organization who contributes data indirectly to an aggregation service (e.g. Europeana)"}],"Campus":[{"label":"Campus","value":"UBCV","attrs":{"lang":"en","ns":"https:\/\/open.library.ubc.ca\/terms#degreeCampus","classmap":"oc:ThesisDescription","property":"oc:degreeCampus"},"iri":"https:\/\/open.library.ubc.ca\/terms#degreeCampus","explain":"UBC Open Collections Metadata Components; Local Field; Identifies the name of the campus from which the graduate completed their degree."}],"Creator":[{"label":"Creator","value":"McMaster, William Robert","attrs":{"lang":"en","ns":"http:\/\/purl.org\/dc\/terms\/creator","classmap":"dpla:SourceResource","property":"dcterms:creator"},"iri":"http:\/\/purl.org\/dc\/terms\/creator","explain":"A Dublin Core Terms Property; An entity primarily responsible for making the resource.; Examples of a Contributor include a person, an organization, or a service."}],"DateAvailable":[{"label":"Date Available","value":"2010-02-08T20:31:53Z","attrs":{"lang":"en","ns":"http:\/\/purl.org\/dc\/terms\/issued","classmap":"edm:WebResource","property":"dcterms:issued"},"iri":"http:\/\/purl.org\/dc\/terms\/issued","explain":"A Dublin Core Terms Property; Date of formal issuance (e.g., publication) of the resource."}],"DateIssued":[{"label":"Date Issued","value":"1976","attrs":{"lang":"en","ns":"http:\/\/purl.org\/dc\/terms\/issued","classmap":"oc:SourceResource","property":"dcterms:issued"},"iri":"http:\/\/purl.org\/dc\/terms\/issued","explain":"A Dublin Core Terms Property; Date of formal issuance (e.g., publication) of the resource."}],"Degree":[{"label":"Degree (Theses)","value":"Master of Science - MSc","attrs":{"lang":"en","ns":"http:\/\/vivoweb.org\/ontology\/core#relatedDegree","classmap":"vivo:ThesisDegree","property":"vivo:relatedDegree"},"iri":"http:\/\/vivoweb.org\/ontology\/core#relatedDegree","explain":"VIVO-ISF Ontology V1.6 Property; The thesis degree; Extended Property specified by UBC, as per https:\/\/wiki.duraspace.org\/display\/VIVO\/Ontology+Editor%27s+Guide"}],"DegreeGrantor":[{"label":"Degree Grantor","value":"University of British Columbia","attrs":{"lang":"en","ns":"https:\/\/open.library.ubc.ca\/terms#degreeGrantor","classmap":"oc:ThesisDescription","property":"oc:degreeGrantor"},"iri":"https:\/\/open.library.ubc.ca\/terms#degreeGrantor","explain":"UBC Open Collections Metadata Components; Local Field; Indicates the institution where thesis was granted."}],"Description":[{"label":"Description","value":"The effect of continual antigenic stimulation on the immune system of mice was studied using two different experimental approaches. A GVHR was induced in Fi mice by the injection of parental spleen cells at weekly intervals. Several weeks later the spleen cells of mice undergoing a GVHR were shown to be immunosuppressed as their in vitro responses to the mitogens Con A and LPS were substancially lower than control animals. The serum from these treated mice was also immunosuppressive to normal spleen cells. The proliferative response to Con A and allogeneic cells of normal: syngeneic, allogeneic, and parental spleen cells was 90% suppressed when GVH serum was added in comparison to the addition of normal serum. Similarly, the in vitro antibody response to a T dependant antigen was impaired; however, the antibody response to a T independant antigen was not impaired. These results indicate that T cell functions are more sensitive than are B cell function to immunosuppressive factors in the serum of mice undergoing a GVHR.\r\nThe serum was fractionated by gel filtration on a Bio-Gel P-200 column. The inhibitory material in GVH serum eluted in the immunoglobulin fraction of serum which indicate that it has a molecular weight of 150,000 or greater.\r\nThe second approach studied involved continual allogeneic stimulation. Parental type mice were injected at five day intervals with Fi spleen cells in order to induce a HVG reaction. After several injections the spleen cells from these mice were tested in vitro. The spleen cells from HVG mice responded the same as normal spleen cells to the mitogens Con A and LPS. The spleen cells from HVG mice showed an enhanced in vitro antibody response as compared to normal spleen cells. This enhancement was attributed to the allogeneic effect.\r\nThis series of experiments have shown that the induction of a GVHR in mice can later lead to immunosuppression and production of immunosuppressive\r\n\r\nfactors in the serum of these mice. The induction of a short term HVG reaction has no adverse effects on the immune system except for enhancing an antibody response. It is possible that a more prolonged HVG reaction would parallel the immunosuppression observed in mice undergoing a GVHR.","attrs":{"lang":"en","ns":"http:\/\/purl.org\/dc\/terms\/description","classmap":"dpla:SourceResource","property":"dcterms:description"},"iri":"http:\/\/purl.org\/dc\/terms\/description","explain":"A Dublin Core Terms Property; An account of the resource.; Description may include but is not limited to: an abstract, a table of contents, a graphical representation, or a free-text account of the resource."}],"DigitalResourceOriginalRecord":[{"label":"Digital Resource Original Record","value":"https:\/\/circle.library.ubc.ca\/rest\/handle\/2429\/19791?expand=metadata","attrs":{"lang":"en","ns":"http:\/\/www.europeana.eu\/schemas\/edm\/aggregatedCHO","classmap":"ore:Aggregation","property":"edm:aggregatedCHO"},"iri":"http:\/\/www.europeana.eu\/schemas\/edm\/aggregatedCHO","explain":"A Europeana Data Model Property; The identifier of the source object, e.g. the Mona Lisa itself. This could be a full linked open date URI or an internal identifier"}],"FullText":[{"label":"Full Text","value":"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 \u00a5 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\u00b0C 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\u00b0C. 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\u00b0C 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\u00b0C. 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\u00b0C 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\u00b0C 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\u00b0C. 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), \u2022 - \u2014 \u2022 ; 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, \u2022\u2014-\u2022; 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, \u2022 \u2022; 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 \u00b1 111 5925 + 420 6743 + 283 2.0 2641 + 634 471 \u00b1 40 6049 + 241 6898 + 641 4.0 695 + 69 76 \u00b1 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. 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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. 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