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The influence of allogeneic or syngeneic cells surface backgrounds on the antibody response of mice to… Acres, Robert Bruce 1977

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THE INFLUENCE OF ALLOGENEIC OR SYNGENEIC CELLS SURFACE BACKGROUNDS ON THE ANTIBODY RESPONSE OF MICE TO RABBIT Fab' FRAGMENTS by ROgERT BRUCE ACRES B.Sc. Un i v e r s i t y of B r i t i s h Columbia, 1974 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In the Department of Microbiology We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February, 1977 Robert Buce Acres, 1977 In presenting th i s thesis in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th i s thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th is thes is for f inanc ia l gain sha l l not be allowed without my writ ten permission. Depa rtment The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 T i ABSTRACT Recent work has shown that i n v i t r o , the cytotoxic immune response to c e l l surface antigens i s enhanced i f the antigen to which the immune response i s d i r e c t e d , i s on c e l l s bearing major h i s t o c o m p a t i b i l i t y a n t i -gens i d e n t i c a l to those of the responding c e l l s . This 'H-2 r e s t r i c t i o n ' has been demonstrated i n the mouse using v i r a l l y infected c e l l s , hap-tenated c e l l s , c e l l s bearing the male Y antigen, and c e l l s d i f f e r i n g at the minor h i s t o c o m p a t i b i l i t y l o c i . Other i n v e s t i g a t i o n s have shown that antigenic determinants coupled to tolerated antigens or isologous serum proteins, e l i c i t a humoral response which i s weaker than that to the same determinant coupled to a heterologous c a r r i e r . This and other evidence suggest an inverse r e l a t i o n s h i p between humoral and c e l l mediated immunity. The purpose of this i n v e s t i g a t i o n was to explore the humoral response to antigens on c e l l s which are syngeneic or allogeneic to the r e c i p i e n t , i n order to determine the influence of a tolerated as opposed to allogeneic background. The approach used i n t h i s study was as follows: Mice were immunized with antigen (rabbit Fab' fragments) attached to syngeneic, a l l o g e n e i c , or F^ (semi syngeneic), i r r a d i a t e d spleen c e l l s . S p e c i f i c a n t i - r a b b i t Fab' plaque forming c e l l numbers were determined f i v e days a f t e r the t h i r d , weekly i n j e c t i o n of Fab' coated spleen c e l l s . Some of the spleen c e l l s taken from the responding animals, on the day of s a c r i f i c e , were incubated i n v i t r o with soluble antigen (rabbit Fab' fragments not s p e c i f i c f o r mouse c e l l s ) f o r four days. The r e s u l t s showed that the humoral response to antigens attached to c e l l s bearing ' s e l f h i s t o -c o m p a t i b i l i t y antigens ( i . e . syngeneic or F 1 semi syngeneic c e l l s ) was s i g n i f i c a n t l y weaker than the humoral response to the same antigen on allogeneic c e l l s . The e f f e c t of i n v i t r o incubation of responder spleen c e l l s f o r four days with soluble antigen was to reverse t h i s d i f f e r e n c e . Those spleen c e l l s e x h i b i t i n g lowered plaque forming c e l l numbers i n i t i a l l y ( i . e . those c e l l s from mice immunized with antigen on syn-geneic or c e l l surfaces) showed, a f t e r incubation, a response equal to or greater than those c e l l s which i n t i a l l y (before i n v i t r o incubation) demonstrated a larger response ( i . e . c e l l s from those mice immunized with antigen on allogeneic c e l l surfaces). i i i TABLE OF CONTENTS Page INTRODUCTION 1 MATERIALS AND METHODS 3 Preparation of the Test Antigens 3 Immunization 4 Chemical Attachment of Antigen to C e l l s . 5 Plaque Assay 6 In V i t r o C e l l Culture 7 Experimental Animals 7 In V i t r o Mitogen and Antigen Stimulation 8 RESULTS 9 In V i t r o Stimulation of Responder Spleen C e l l s 3 ' Measured by H Thymidine Uptake 16 DISCUSSION . 17 BIBLIOGRAPHY 20 LIST OF TABLES Immune response of C57B1/6 mice to rabbit Fab' fragments presented on DBA/2, C57B1/6 or B..D F D 2 I spleen c e l l s Immune response of CBA/J mice to rabbit Fab' on C57B1/6, DBA/2 or CBA/J spleen c e l l s Immune response of C57B1/6 mice to l O i j g Rabbit Fab' mixed with ei t h e r C57B1/6 or DBA/2 spleen c e l l s E f f e c t of i n v i t r o c u l t u r i n g of spleen c e l l s from mice previously immunized with R.Fab' coated, syngeneic or allogeneic c e l l s . V LIST OF FIGURES Page Figure 1 Time course study of the immune response of 10 C57B1/6 mice immunized at weekly i n t e r v a l s with 10^, Fab' coated, i r r a d i a t e d spleen c e l l s of syngeneic or allogeneic o r i g i n ACKNOWLEDGEMENT I would l i k e to thank Dr. J u l i a Levy for her encouragement, suggestions, and unending patience during the research f o r , and preparation of t h i s t h e s i s . ABBREVIATIONS Abbreviations used i n t h i s thesis are: MHC major h i s t o c o m p a t i b i l i t y complex H-2 that region of the mouse genome encoding the MHC antigens and the immune response genes BSA bovine serum albumin Fab' approximately 1/3 of the immunoglobulin molecule which does not f i x complement and contains 1 antigen binding s i t e CFA complete Freund's adjuvant PBS phosphate buffered s a l i n e DEAE d i e t h y l amino e t h y l SRBC sheep red blood c e l l s PFC plaque forming c e l l s DPM d i s i n t e g r a t i o n s per minute HBSS Hank's balanced s a l t s o l u t i o n EDCI l-Ethyl-3-(3-Dimethylaminopropyl) Carbodimide LPS lipopolysaccharide 3 H Tritxum CPM counts per minute 1 INTRODUCTION The r o l e of the genes of the major h i s t o c o m p a t i b i l i t y complex (MHC) of mice i n the generation of various immunological responses has been studied i n t e n s i v e l y i n the past few years. I t has been shown that co-operation between B and T c e l l s occurs only when they share at l e a s t one set of H-2 antigens (1-4). More recently, i t has been shown that i n order for v i r u s immune T c e l l s to generate an i n v i t r o cytotoxic reaction to v i r a l l y i n f e c t e d c e l l s , i t i s necessary for the e f f e c t o r and target c e l l s to share e i t h e r the K or D regions of the MHC (5-7). This l a t t e r aspect of H-2 r e s t r i c t i o n also applies to chemically modified (haptenated) target c e l l s (8-10), those e x h i b i t i n g minor h i s t o c o m p a t i b i l i t y antigens (11), or the male Y antigen (12). A l l of these observations imply the importance of recognition of H-2 gene products i n the i n t e r a c t i o n of lymphocytes and target c e l l s i n the generation of cytotoxic T c e l l s . Recent observations of T r i n c h i e r i jet al (13) however, in d i c a t e there may be an inverse r e l a t i o n s h i p between the generation of cytotoxic T c e l l s and a humoral response to v i r u s antigens. Using SV-40 transformed human-mouse fusion c e l l products, these i n v e s t i g a t o r s found that while H-2 r e s t r i c t i o n of cell-mediated immunity prevailed, the a n t i - v i r a l antibody t i t r e was highest i n those mice not showing the presence of c e l l s cyto-toxic for SV-40 transformed human c e l l s . This inverse r e l a t i o n s h i p has also been demonstrated with such antigens as tuberculin (Janichi et; a l 1970), lysozyme (Thompson et a l 1972) and chemically modified antigens (Parish 1972). That the humoral response to antigens may be i n h i b i t e d or suppressed by the a s s o c i a t i o n of the antigen with tolerated antigens has been demon-strated with a number of systems. S c i b i e n s k i ejt a l (14) reported that 2 mice, neonatally t o l e r i z e d to lysozyme and subsequently challenged with lysozyme covalently linked to bovine serum albumin (BSA), showed a markedly suppressed response to BSA when compared to the response of lysozyme tolerant animals immunized with a mixture of BSA and lysozyme (not li n k e d ) . S i m i l a r l y , Lee and Sehon (15) found that mice challenged with haptenated, s e l f serum proteins (epitope density of 9, 11 or 12 haptens per c a r r i e r molecule) showed a suppressed anti-hapten response when compared to the response i n animals immunized with the hapten conjugated to a foreign immunogenic prot e i n . These apparently unrelated observations r a i s e some i n t e r e s t i n g questions regarding the r e l a t i o n s h i p of humoral vs. cell-mediated immune responses, and the r o l e of s e l f or non-self markers on c e l l s with which new or non-self antigens are associated. The work reported i n t h i s thesis was designed to investigate t h i s r e l a t i o n s h i p . E s s e n t i a l l y , I studied the humoral response i n mice to an antigen (rabbit Fab' fragments) which was presented during immunization i n asso c i a t i o n with i r r a d i a t e d lymphoid c e l l s of syngeneic, allogeneic or F^ o r i g i n . Since H-2 re-s t r i c t i o n , as measured so f a r , has only involved studies on the generation of cytotoxic T c e l l s , i t was f e l t that an analogous study on the generation of helper T c e l l s and antibody forming c e l l s would help to complete the understanding of the r o l e of MHC i n immune responsiveness. An under-standing of the influence of self-vs.-non-self backgrounds on immune responsiveness to membrane associated antigens of either v i r a l and/or neoplastic o r i g i n s may have broad s i g n i f i c a n c e i n a number of disease conditions. 3 MATERIALS AND METHODS Preparation of the Test Antigens Rabbit Fab' fragments (R.Fah') with s p e c i f i c i t y for e i t h e r sheep erythrocytes (SRBC) or c e l l surface antigens of lymphocytes from various mouse s t r a i n s (C57 B1/6J, DBA/2J and CBA/J) were prepared as follows: A 50% suspension of SRBC i n phosphate buffered s a l i n e (PBS) was emulsified with 25% (v/v) of Complete Freund's Adjuvant (CFA). A t o t a l volume of 1.0 ml was i n j e c t e d i n 4 s i t e s Intramuscularly, and one i n t r a p e r i t o n e a l l y into r a b b i t s . Animals were boosted at monthly i n t e r v a l s with 1.0 ml of 50% SRBC i n PBS administered at f i r s t intravenously then intramuscularly, and bled one week a f t e r i n j e c t i o n . I n i t i a l immunization with splenic g mouse lymphocytes was with 10 c e l l s i n PBS and 25% CFA administered g as above. Subsequent immunizations were c a r r i e d out with 10 lymphocytes i n PBS given intramuscularly at monthly i n t e r v a l s with blood being taken 1 week following i n j e c t i o n . The immunoglobulin f r a c t i o n of the immune serum was prepared according to standard methods with ammonium s u l f a t e p r e c i p i t a t i o n (40% saturation) followed by d i a l y s i s and ion exchange chromatography on DEAE c e l l u l o s e (16). Fab' fragments were prepared by papain di g e s t i o n according to the method of Porter (17). The Fab' fragments with s p e c i f i c i t y f o r mouse c e l l surface antigens was used to bind to i r r a d i a t e d mouse lymphocytes which were subsequently used for immunization of r e c i p i e n t s . The Fab' fragments s p e c i f i c for SRBC were used to coat these c e l l s f o r use i n the plaque assay. 4 Immunization Mouse splenic lymphocytes to be used f o r immunization were prepared by passage of clumps of c e l l s through a s t a i n l e s s s t e e l 50 gauge mesh i n PBS containing 5% f o e t a l c a l f serum (FCS). The c e l l s were treated with .83% NH^Cl i n 5% FCS for 4 min and centrifuged at 200 x g to remove erythrocytes, and washed twice i n PBS containing 5% FCS. These c e l l s were i r r a d i a t e d with 2000 rads on a Gammacell 220, Atomic Energy of Canada Ltd., and were incubated with appropriate s p e c i f i c R.Fab' fragments at a concentration of 0.5 mg per lO'' spleen c e l l s per ml i n Hepes buffered 1640 medium (Grand Island B i o l o g i c a l Co.) containing 5% FCS. The coated c e l l s were washed three times i n s a l i n e and suspended at a concentration g of 10 c e l l s per ml. C e l l s were counted by the trypan blue exclusion method. Recipient mice were i n j e c t e d i n t r a p e r i t o n e a l l y with 10^ of the R.Fab' coated lymphocytes at weekly i n t e r v a l s , s a c r i f i c e d 5 days a f t e r the l a s t i n j e c t i o n , and t h e i r spleens used for assay of plaque forming c e l l s (PFC) using R.Fab' coated SRBC as the antigen. The amount of R.Fab' attached to the immunizing mouse lymphocytes was quantitated as follows. An al i q u o t of the R.Fab' fragments to be used was iodinated with 1 2 5 i by the chloramine T method (18) and the s p e c i f i c a c t i v i t y determined per mg protein, using CPM measured on a Beckman Biogamma counter and an e x t i n c t i o n c o e f f i c i e n t f o r the Fab' fragments as O.D.^gQ 1.5 mg protein ml Known proportions of tracer 1 2 5 I l a b e l l e d R.Fab' were admixed with unlabelled R.Fab' during the i n -cubation with the i r r a d i a t e d lymphocytes. Subsequently, the amount of r a d i o a c t i v i t y recovered on the l a b e l l e d c e l l s was assumed to be proportional to the r e l a t i v e amount of unlabelled R.Fab' fragments attached. In a l l 5 instances, regardless of the s t r a i n of mouse lymphocytes used, i t was found that 10 Jig of Fab' + 10% was bound to 10^ lymphocytes. Thus i t was assumed that each i n j e c t i o n of R.Fab' coated c e l l s Into r e c i p i e n t mice constituted immunization with 10.0 yg of R.Fab' fragments. Mouse kidney c e l l s were o r i g i n a l l y used f o r immunization, but, due to poor c e l l s u r v i v a l and tedious i s o l a t i o n procedures, they were abandoned i n favour of h e a r t i e r and more e a s i l y obtainable spleen c e l l s . I s o l a t i o n of kidney c e l l s was c a r r i e d out as follows: Mouse kidneys were cut into small pieces and suspended i n prewarmed (37°) 0.25% t r y p s i n i n Hank's balanced s a l t s o l u t i o n (8 to 10 kidneys to 50 ml of HBBS). This suspension was s t i r r e d for 1 hour at 37°, the large pieces allowed to s e t t l e , and the supernatant containing the c e l l s pipetted o f f . The large pieces were then resuspended i n fresh 0.25% t r y p s i n i n HBBS and the procedure repeated, u n t i l only white connective tissue was l e f t i n the suspension. The c e l l s i n the supernatant were centrifuged and washed i n PBS, treated with 0.83% NH^Cl for 3 minutes, to remove erythrocytes, washed again, and layered on top of 8 ml FCS and centrifuged at 500 rpm i n order to remove pieces of kidney s t i l l remaining. S u r v i v a l rate was roughly 10"* c e l l s per kidney. Chemical Attachment of Antigen to C e l l s 1. Coupling with l-Ethyl-3-(3-Dimethylaminopropyl) Carbodimide HC1 (EDCI): Rabbit Fab' fragments prepared from rabbits immunized with SRBC were attached to mouse kidney or spleen c e l l s with EDCI. Rabbit red blood c e l l s were used to determine which concentration of EDCI would 6 give maximum coupling with the least c e l l damage. Maximum attachment with the least l y s i s was obtained with 12 mg per ml EDCI and 1 mg per ml Fab'. However, i t was discovered that after attachment of Fab' to spleen cells with EDCI, and washing with PBS, most cells absorbed trypan blue, indicating c e l l death, and that those cells which did survive were some-what rough around the edges. Also, i t was shown that EDCI is a hapten i t s e l f (Dale Gregerson, personal communication). For these reasons, the method of attachment was abandoned. Other chemical fixatives which were tested include: Glutaraldehyde, which l e f t c ells alive and intact, but attached Fab' poorly; and several maleimides, a l l of which were insoluble in physiological saline. Plaque Assay Immunized animals were sacrificed 5 days after their last injection and their spleen cells used to assay the number of antibody forming cells (AFC) capable of responding to R.Fab' fragments. The microscope slide assay described by Cunningham (19) was used. SRBC were coated with anti-SRBC R.Fab' fragments by incubation of a 25% suspension of SRBC in PBS with the R.Fab' fragments at a concentration of 0.5 mg per ml at 37° for 1 h. The coated SRBC were washed three times and used in the assay. PFC assays for IgG-producing cells from each mouse were run in triplicate with the coated SRBC and SRBC alone. The number of specific anti-rabbit Fab' PFC was calculated by subtracting the background plaques found with SRBC alone, from the number with the R.Fab' coated SRBC. The reaction mixture for the PFC assay contained the following materials: 0.3 ml of Hepes buffered 1640 medium plus 5% FCS containing 5 x 10^ mouse spleen lymphocytes, 0.1 ml of 20% SRBC (coated or uncoated), 0.05 ml of guinea 7 pig serum as a source of complement (the serum had been previously absorbed with R.Fab 1 coated SRBC and mouse spleen lymphocytes), and 0.05 ml of a 1/20 d i l u t i o n of r a b b i t anti-mouse Ig serum. Each plaque chamber holds 0.1 ml of mixture, therefore 10^ mouse spleen c e l l s . Plaques were allowed to develop at 37° for 1% h. In v i t r o C e l l Culture Aliquots of spleen lymphocytes, taken from immunized mice 5 days a f t e r t h e i r l a s t i n j e c t i o n and assayed immediately for PFC, were also cultured i n v i t r o and tested f o r the presence of PFC. Culturing procedures have been described previously (20). B r i e f l y , 1 x 10^ lymphocytes were cultured i n 35 mm P e t r i dishes (Falcon No. 3301) i n RPMI 1640 medium containing 10% FCS ( M i c r o b i o l o g i c a l Associates No. 84557, Bethesda, Md.) and 5 x 10 ~* M 2-mercaptoethanol. C e l l s were cultured for 4 days i n the presence of varying concentrations of R.Fab'. When they were harvested, they were tested f or the presence of PFC for R.Fab' fragments as described above. Experimental Animals Female mice of the s t r a i n s C57B1/6J, DBA/2J, CBA/J and C57B1/6J x DBA/2J F^ (BgD^F^) between 2 and 4 months of age were used i n the following experiments. They were obtained from the Jackson Laboratories (Bar Harbor, Maine). Outbred albino rabbits were used for the preparation of Fab' f r a g -ments of various s p e c i f i c i t i e s . 8 In vitro Mitogen and Antigen Stimulation Spleen cells were prepared in phosphate buffered saline (PBS) + 5% • fetal calf serum (FCS). The cells were centrifuged at 200 x g for 5 minutes, resuspended in PBS + 5% FCS, and the v i a b i l i t y determined by trypan blue exclusion. Desired numbers of cells were centrifuged and resuspended in RPM1 1640 and dispensed into microtitre plates (Linbro Chemical Co., New Haven, Conn.). Each well contained 5 x 10^ viable spleen cells in a f i n a l volume of 0.25 ml medium with FCS at a f i n a l concentration of 2%, and 5 yg concanavalin A, 6.25 yg lipopolysaccharide, or a range of concentrations of soluble rabbit Fab'. The cultures were incubated in a humidified atmosphere of 95% ai r - 5% CO^ for 4 days. 3 Eighteen hours before the cells were harvested, 1.0 yCi of H-thymidine (specific activity 5.0 Ci/mmole, New England Nuclear, Montreal, Canada) was added to each well. The cells were harvested onto glass fibre f i l t e r paper, dried, and the radioactivity determined on a Nuclear Chicago Unilux II s c i n t i l l a t i o n counter. RESULTS The time course of the response of C57 Bl/6 mice immunized with R.Fab fragments attached to ei t h e r syngeneic or allogeneic (DBA/2J) i r r a d i a t e d lymphocytes i s shown i n F i g . 1. I t can be seen that the response at a l l times tested, of those mice immunized with R.Fab' fragments attached to allogeneic c e l l s was higher i n terms of PFC, and a f t e r the t h i r d i n j e c t i o n the differences were s i g n i f i c a n t . Because the responses were maximal a f t e r the t h i r d i n j e c t i o n subsequent experiments were c a r r i e d out at t h i s time. In order to determine the r e p r o d u c i b i l i t y of t h i s observation and to observe the e f f e c t of a mixture of allogeneic and syngeneic c e l l markers on the immunizing c e l l s , several experiments were performed i n C57B1/6 mice Immunized three times with R.Fab' l a b e l l e d lymphocytes from syngeneic (C57B1/6), allogeneic (DBA/2J) and F ( B 6 D 2 F ] _ ) animals. The r e s u l t s i n Table 1 show that the responses of animals immunized with both syngeneic and F^ l a b e l l e d c e l l s were s i g n i f i c a n t l y lower than those of mice immunized with allogeneic l a b e l l e d c e l l s . I f t h i s d i f f e r e n c e were a t t r i b u t a b l e to the previously observed allogeneic e f f e c t (21), one might expect that the anti-SRBC response i n the PFC controls would be higher and that the F^ c e l l s would generate a s i m i l a r e f f e c t . This does not appear to be the case (Table 1). It would appear, rather, that the presence of ' s e l f antigens on the l a b e l l e d c e l l s has a negative e f f e c t on the generation of PFC to R.Fab' fragments. In order to determine whether t h i s observation was s t r a i n s p e c i f i c , another seri e s of experiments was run with CBA mice immunized with ei t h e r syngeneic (CBA) or allogeneic (DBA or C57B1/6) lymphocytes l a b e l l e d with R.Fab' fragments. Analogous r e s u l t s were obtained i n these instances (Table 2). 10 CO NUMBER O F I N J E C T I O N S Figure 1 Time-course study of C57B1/6J mice immunized at weekly i n t e r v a l s with 10^, Fab' coated, i r r a d i a t e d spleen c e l l s of C57B1/6J or DBA/2J o r i g i n . Recipient spleen c e l l s were assayed for antl-R.Fab' PFC 5 days a f t e r each weekly i n j e c t i o n . Results are expressed as anti-R.Fab 1 PFC per 10^ r e c i p i e n t spleen c e l l s and represent the net t o t a l of anti-Fab' plaques a f t e r subtraction of spontaneous anti-SRBC plaques. Each point represents the average of 6 mice. O Q R.Fab' coated C57B1/6J c e l l s i n j e c t e d ® © R.Fab' coated DBA/2J c e l l s i n j e c t e d Table 1: Immune response of C57B1/6 mice to Rabbit Fab' presented on DBA/2J, C57B1/6 or B,,D„F., c e l l s . A l l numbers represent PFC o 2. 1 per spleen + S.E.M. P = <0.02 according to Student's t test, (combined allogeneic vs. combined syngeneic and combined allogeneic vs. combined F^). Each number represents the average of 3 mice. Experiment R.Fab' coated A n t i R.Fab' PFC/spleen Anti-SRBC PFC/spleen c e l l s i n j e c t e d 1 DBA/2J 24675 + 8937 177 + 57 C57B1/6 5298 + 1744 150 + 46 B 6 D 2 F 1 5458 + 1914 216 + 21 2 DBA/2J 11611 + 2231 63 + 62 C57B1/6 4201 + 1442 33 + 17 B 6 D 2 F 1 1110 + 462 96 + 27 3 DBA/2J 10203 + 4415 275 + 126 C57B1/6 6891 + 265 1126 + 609 B 6 D 2 F 1 4896 + 4577 265 + 93 12 Table 2: Immune response of CBA/J mice to R.Fab' on C57B1/6, DBA/2J or CBA/J spleen c e l l s . Results are expressed as PFC per spleen ± S.E.M. P = <0.01 according to Student's t test (combined allogeneic vs. combined syngeneic). Each number represents the average of 3 mice. Experiment R.Fab' coated cells Anti R.Fab' PFC/spleen injected ± S.E.M. 1 C57B1/6 36395 + 1429 CBA/J 16198 + 1119 2 DBA/2J 33395 + 8976 CBA/J 15754 + 193 The p o s s i b i l i t y that these differences i n response to R.Fab' fragments might simply be an e f f e c t of asso c i a t i o n of these molecules with ei t h e r syngeneic or allogeneic c e l l s rather than being a r e s u l t of the Fab' fragments being attached to the c e l l surfaces was also examined. Fab' fragments with no s p e c i f i c i t y f o r mouse c e l l surface antigens were prepared and mixed with e i t h e r syngeneic or allogeneic c e l l s at concentrations equivalent to those used with l a b e l l e d c e l l s . The time course of the responses of C57B1/6J mice immunized with e i t h e r C57B1/6J or DBA i r r a d i a t e d spleen lymphocytes mixed with 10 yg Fab' was followed (Table 3). I t can be seen that no s i g n i f i c a n t differences occurred between the 2 groups of immunized mice. Further studies were c a r r i e d out to determine the e f f e c t of i n v i t r o culture of immune spleen lymphocytes i n the presence of varying concen-t r a t i o n s of R.Fab'. Over a range of 2.0 to 30.0 yg of Fab' per culture, Immune lymphocytes responded s p e c i f i c a l l y to the R.Fab' fragments. A dose l e v e l of 10 yg Fab' per culture was established since i t appeared to be close to optimal l e v e l s and was equivalent to the i n vivo stimulus. A s e r i e s of experiments were performed using C57B1/6 or CBA mice as r e c i p i e n t s of l a b e l l e d syngeneic, allogeneic or F^ lymphocytes. The PFC per 10 7 c e l l s was determined at the time of s a c r i f i c e (5 days a f t e r the f i n a l immunization) and aliquots of spleen c e l l s were subsequently cultured with 10 yg R.Fab' for 4 days, harvested and assayed f o r PFC with R.Fab' coated SRBC. The r e s u l t s are summarized i n Table 4. I t can be seen that while the PFC i n r e c i p i e n t s of eit h e r syngeneic or F^ l a b e l l e d c e l l s was depressed at the time of s a c r i f i c e i n comparison to r e c i p i e n t s of allogeneic l a b e l l e d c e l l s , the PFC to R.Fab' fragments i n the former group, a f t e r i n v i t r o culture was equal to or greater than those observed i n the l a t t e r group. Table 3: Immune response of C57B1/6 mice to 10 Ug R.Fab' mixed with either C57B1/6 or DBA/2J irradiated splenic lymphocytes (R.Fab' not specific for mouse c e l l s ) . Results are expressed as PFC per 10 host spleen cells ± S.E.M. Each number represents the average of' 2 mice. Injection Cells Injected (mixed with 10 yg R.Fab') PFC per 10 7 responding 2° DBA 11.5 ± 1.5 C57 1.5 ± 1.5 3° DBA 65 ± 45 C57 80 ± 57 4° DBA 177 ± 100 C57 325 ± 74 5° DBA 136 ± 30 C57 162 ± 12 6° DBA 18 ± 18 C57 42 ± 6 15 Table 4: Effect of in vitro culturing of spleen cells from mice previously immunized with R.Fab' coated lymphocytes of syngeneic or allogeneic origin. Culture medium contained 10 yg R.Fab' (non-specific for mouse cells) per 10 7 cultured ce l l s . PFC are presented as per 10 7 recipient spleen cells at the time of sacrifice (Day 0) and after 4 days in culture with 10 yg free R.Fab'. Experiment Recipient Fab' coated PFC/10 PFC/10 cells injected spleen cells spleen cells DAY 0 DAY 4 1 C57 DBA 2280 + 618 946 ± 116 C57 C57 448 + 204 1329 ± 544 C57 • B6 D2 F1 478 + 203 1674 ± 395 2 C57 DBA 995 + 346 233 ± 28 C57 C57 575 + 105 462 ± 137 C57 B6 D2 F1 750 + 662 404 ± 76 3 CBA C57 2885 + 567 438 ± 148 CBA CBA 1275 + 125 642 ± 305 4 CBA C57 996 + 154 233 ± 31 CBA CBA 282 + 2 535 ± 119 5 CBA DBA 875 + 450 248 ± 78 CBA CBA 341 + 73 352 ± 124 16 3 In v i t r o Stimulation of Responder Spleen C e l l s Measured by H Thymidine  Uptake. 3 Increased uptake of H-thymidine by immune lymphocytes cultured i n the presence of appropriate concentrations of antigen i s thought to be mainly an i n d i c a t i o n of T c e l l responsiveness. For th i s reason i t was thought that comparative data could be obtained f o r T c e l l s from mice s e n s i t i z e d to R.Fab' on syngeneic vs. allogeneic backgrounds. Under no circumstances i n the course of these studies could s i g n i f i c a n t l e v e l s of stimulation of immune lymphoid c e l l s cultured i n the presence of R.Fab' be achieved, regardless of the means of immunization. Mitogen stimulation of these c e l l s by Concanavalin A or LPS i n culture was normal. DISCUSSION Considerable evidence e x i s t s that indicates immune homeostasis i s the r e s u l t of a balance between responsiveness and suppression, and that non responsiveness to s e l f antigens i s the r e s u l t of active suppression (22-28). Evidence presented by S c i b i e n s k i et a l (14) ( i . e . non responsive-ness to an antigen linked to a tolerogen) suggests active suppression against the tolerogen, which extends to a new antigen, coupled to that tolerogen. Lee and Sehon (15) using haptenated mouse immunoglobulin, showed that the suppression to the hapten i s maintained during adoptive transfer experiments, implying the presence of suppressive c e l l s , whose suppression extends to new determinants coupled to the tolerated antigen. Humoral a u t o r e a c t i v i t y has, i n the past, been enhanced by the administration of LPS (29) or rab b i t anti-mouse thymocyte serum (30-22), the explanation i n the l a t t e r case, being the removal of suppressive T c e l l s . Although conclusive evidence for the presence of c e l l s which suppress a u t o r e a c t i v i t y i s not presented i n t h i s study, the data support such a theory. Rabbit Fab' fragments, when attached to syngeneic or semi syngeneic (F^) c e l l s , generate a decreased humoral immune response, when compared to the response mounted against the same antigen presented on allogeneic c e l l s . This observation appears to hold regardless of the s t r a i n combinations used. If non responsiveness to s e l f i s maintained by suppressor c e l l s , then, as was the case i n Scibienski's work, t h i s non responsiveness includes antigens which are attached to the tolerated antigen (in t h i s case s e l f , c e l l surface antigens). I f , on the other hand, the response toward R.Fab' on syngeneic c e l l s was not suppressed, but the response towards R.Fab' on allogeneic c e l l s was enhanced, due to the presence of alloantigens, one would also expect an enhanced response to the same antigen on any c e l l s bearing allogeneic antigens. This i s not the case, the response towards R.Fab' on semi syngeneic (also semi allogeneic) F^ c e l l s was as low as that towards R.Fab' on syngeneic c e l l s . Thus, I t would appear to be the presence of s e l f antigens which cause the decreased response, rather than the presence of allogeneic antigens which enhance the response. The observation that background anti-SRBC plaques i n our assays were never markedly d i f f e r e n t between experimental groups would i n d i c a t e that i n the conditions used here, no marked all o g e n e i c e f f e c t was taking place. Abrogation of s e l f tolerance i n v i t r o has i n the past, been accom-plished by incubating immune competent c e l l s with s e l f c e l l s (25, 26), with LPS (31, 32) or by incubation i n culture medium alone (33). These findings are consistent with the r e s u l t s obtained i n t h i s study: those spleen c e l l s which showed a low a n t i R.Fab' response on day 0 (Table 4), i . e . spleen c e l l s from those mice r e c e i v i n g R.Fab' on syngeneic c e l l s , a f t e r 4 days jLn v i t r o Incubation with soluble antigen, showed a response equal to or larger than spleen c e l l s from those mice re c e i v i n g R.Fab' on allogeneic c e l l s . This suggests the presence i n immunized animals of p o t e n t i a l l y reactive c e l l s , but that t h e i r development into antibody producing c e l l s i s i n some way i n h i b i t e d i n vivo. Suppression of the humoral response against heterologous antigens on the surface of ' s e l f c e l l s , seems an i n v i t i n g explanation f o r the weak immune r e a c t i v i t y seen toward tumour antigens i n many instances. Recent work i n animal systems (tumours and v i r a l l y i n f e c t e d c e l l s ) has indicated that potent c e l l mediated immunity, assayed by the i n v i t r o generation of cytotoxic c e l l s , can be developed against syngeneic c e l l s demonstrating al t e r e d a n t i g e n i c i t y . Indeed, these cytotoxic c e l l s are only able to d e l i v e r the k i l l i n g s i g n a l to those modified target c e l l s sharing at l e a s t one of the major h i s t o c o m p a t i b i l i t y antigens (5, 11, 8). These observations would appear to be i n contrast to those reported here. However, i t should be pointed out that while marked c y t o t o x i c i t y can be measured i n mice against t h e i r syngeneic tumors, t h i s response appears to be under r a p i d l y developing stringent and s p e c i f i c suppression mediated by T c e l l s (34). Whether or not the lowered humoral response seen i n animals immunized with an antigen linked to syngeneic c e l l s i s a manifestation of s p e c i f i c suppression of s e l f or s e l f - a s s o c i a t e d c e l l surface markers remains to be c l a r i f i e d . I t i s , however, c l e a r that the a s s o c i a t i o n of an antigen with syngeneic, as opposed to allogeneic c e l l surface antigens does have a s i g n i f i c a n t e f f e c t on the humoral response to that antigen. BIBLIOGRAPHY Kindred, B. and Shreffler, D.C. 1973. H-2 dependence of cooperation between T and B cells in vivo. J. Immunol. 109: 940. 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