A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE

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1 SECOND SYMMETRY IN THE Balb/c-C57BL/6 (B6) SYSTEM, AND ANTI-ANTI-SELF ANTIBODIES IN OLD B6 MICE By ABDULAZIZ FAHEEMI B.Sc, Eastern Washington University, 1989 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Microbiology & Immunology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March 1994 Abdulaziz Faheemi, 1994

2 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. (Signature) Department of Microbiology and Immunology The University of British Columbia Vancouver, Canada Date March 22, 1994 DE-6 (2/88)

3 ii Abstract In order to establish the presence of anti-anti-self antibodies and study such MHC mimicking antibodies in the sera of alloimmune mice, reciprocal sets of alloantisera were raised between the following pairs of mice : Balb/c and C57BL/6 (B6), Balb/c and CBA, and B6 and CBA. After 10 weeks of alloimmunization, the presence of anti-anti-self antibodies could be detected by an avidin-biotin ELISA, and by inhibition of antibody and complement mediated cytotoxicity in the alloimmune B6 and Balb/c sera. Such antibodies belonged to the immunoglobulin G class. Considerably higher levels of anti-antiself antibodies were induced in alloimmune B6 mice compared to alloimmune Balb/c mice. Moreover, anti-anti-self antibodies spontaneously occurred in the serum of old B6 mice. Anti-anti-self antibodies in alloimmune B6 mice were shown to be highly specific. Anti-anti-self antibodies in both alloimmune and old B6 mice specifically inhibited the cytotoxicity of anti-foreign MHC antibodies present in the converse Balb/c anit-b6 antiserum. This indicates that these antibodies have a self MHC mimicking activity.

4 iii Table of Contents Abstract. Table of Contents list of Figures list of Abbreviations Acknowledgment ji. iii. iv..vi..vii Introduction 1. Materials and Methods 10. Results 16. Discussion. 42. Bibliography.49.

5 IV List of Figures Figure Title page 1. The immunization of mice with allogeneic lymphocytes produces anti-foreign and anti-anti-self MHC responses 7. 2a. Binding of biotinylated Balb/c anti-b6 to B6 anti-balb/c sera in the second symmetry ELISA 18. 2b. Biotinylated Balb/c anti-b6 binds to 1, and 1.5 years old B6sera 20. 2c. Binding of biotinylated Balb/c anti-b6 to individual alloimmune and old B6 serum 22. 2d. Balb/c anti-b6 binds to it's reciprocal B6 anti-balb/c and not to any other alloantisera or syngeneic sera Biotinylated B6 anti-balb/c binds to Balb/c anti-b Anti-anti-self antibodies in alloimmune, 1 year and 1.5 year old B6 using affinity purified polyclonal IgG detected by the second symmetry ELISA Anti-anti-self antibodies in alloimmune Balb/c using affinity purified sera IgG Purity of the affinity purified IgG from the mouse serum established by 7.5% SDS-PAGE Cytotoxicity of Balb/c anti-b6 antisera against B6 target 34. cells. 8. Cytotoxicity of B6 anti-balb/c antisera against Balb/c 34. target cells.

6 V Continued List of Figures Figure Tide Page 9. Absence of anti-anti-self antibodies in alloimmune Balb/c sera as detected by the inhibition of cytotoxicity assay Anti-anti-self antibodies in alloimmune and old B6 sera detected by the inhibition of cytotoxicity assay 40.

7 vi List of Abbreviations Abbreviations A405nm AAI Abs AIDS B6 Balb/c CBA CFA CTL ELISA HIV Id(s) Ig kd KLH PAGE PBS SAS SDS SRBC TMA Tris V W/V Meaning. absorbance at 405 nm. auto-anti-idiotype(s). antibodies. acquired immunodeficiency syndrome. C57BL/6 mouse strain. Balb/c mouse strain. CBA mouse strain. complete Freund's adjuvant. cytotoxic T lymphocytes. Enzyme Linked hnmunosorbant Assay..human immunodeficiency virus. idiotype(s). immunoglobulin. Jdlodalton. keyhole limpet hemocyanin. polyacrylamide gel electrophoresis. phosphate buffered saline. saturated ammonium sulphate. sodium dodecyl sulphate. sheep red blood cells. p-anmionphenyl-n-trimethylammonium chloride. tris(hydroxymethyl)aminomethane..variable. weight to volume.

8 Acknowledgments I would like to express my thanks and appreciation to my supervisor, Dr. Geoffrey Hoffmann for his guidance, encouragement, and patience throughout the course of this thesis. I also like to thank the members of my thesis committee, Dr. Michael Gold and Dr. Pauline Johnson for their direction and advice. I am also grateful to the past and present members of the Hoffmann's laboratory, Robert Forsyth, Dr. Michael Grant, Dr. Tracy Kion, Mark Whaley and Dr. Yu- Zhou Yang for their technical advice and stimulating discussions. I also wish to express personal gratitude to my family, and especially my brother A. Majeed for offering encouragement and understanding at all the right moments. Financial support of the United Arab Emirates University is greatly appreciated.

9 1 Introduction The immune system must be able to regulate itself. A dysfunction in immunoregulation may lead to the development of an autoimmune disease, cancer, or failure to eliminate a disease causing agent. The mechanisms of immunoregulation are not well understood. Some of the proposed mechanisms of immunoregulation are: 1) the limited life span of effector cells, 2) antigen removal, 3) antibody feedback, 4) suppressor cells, and 5) the idiotypic network 1. The fifth and most intriguing way the immune response may be regulated, is the idiotypic network, as proposed by Niels Jerne in 19742,3 # The idiotypic network concept emerged from several important observations and is based on two functions of the antibody and antigen binding receptors on lymphocytes molecules: its traditional role in binding antigen, and its ability to be an antigen. Based on Jerne's network hypothesis 2, when animals are immunized with antigen-x, the concentration of anti-x antibody is greatly increased. This increase in anti-x antibody is then perceived by the immune system, and antibody reactive to the idiotypic determinants of anti-x antibody may be generated. The antiidiotypic antibody can then elicit the production of another wave of antibody. Thus, antigen X stimulates anti-x antibody (Abl) that stimulates anti-(anti-x antibody) antibody (Ab2) that stimulates anti-[anti-(anti-x antibody)] antibody (Ab3), and so on. Each response wave, however, is dampened by anti-idiotypic regulatory mechanisms. Jerne's network theory gives the immune system the ability to regulate itself in both positive and negative ways using

10 only itself. Since some antibodies are made even in the absence of foreign antigens a vast number of immune responses are always going on. These internal reflections, a web of opposing immune responses, are in dynamic equilibrium. According to Jerne what we perceive as an immune response when foreign antigen is introduced is simply the perturbation of the pre-existing network and the establishment of new position of equilibrium 2. Furthermore, Jerne postulated that the idiotypic determinants may be thought of as the "internal images" of the external antigens 2. In other words antibody against an antigen binding site "looks like" the corresponding antigen 2. It was therefore predicted that priming an animal with anti-id rather than with an antigen would be possible, and this, in fact, has been successfully achieved in the induction of antibody responses against parasites 4, bacteria 5, and viruses 6. Another interesting property of antiidiotypic antibodies is their use as probes to identify cellular receptors 7. Such approaches have been used to characterize both hormones 7 and viral surface receptors 8. Hoffmann's symmetrical network theory 9 ' 10 ' 1!> 12 is based on Jerne's network theory. However, it also takes into account a considerable range of experimentally observed phenomena, including memory, suppressor and helper T cells, and specific T cell factors. An important part of this theory is the ability of the network to select the clones that are images of particular antigens, including self antigens. The potential importance of regulatory idiotypic interactions is supported by several studies, an illustrative example of which is the

11 study done by Urbain et. al 13. In their experiments they were able to obtain a series of complementary anti-idiotypic antibodies (i.e., Ab2, and Ab3) in the rabbit. In this study, the first rabbit was immunized with Micrococcus lysodikiticus polysaccharide (CHO), and the anti- CHO antibodies from this rabbit were used in a second unrelated rabbit in order to prepare anti-idiotypic antibodies (i.e. Ab2). The Ab2 produced by the second rabbit was used as immunogen to prepare anti-(anti-idiotypic) antibodies (i.e. Ab3) in other unrelated rabbits. Finally Wikler et. al. 14 used Ab3 to prepare in a fourth unrelated rabbit Ab4. The series of anti-idiotypic antibodies obtained in these experiments seems to be true anti-idiotypic antibodies, since they have been prepared in allotype matched rabbits. The immunochemical analysis of Abl-Ab4 suggested that Ab3 shares idiotypic determinants with anti-cho antibodies (Abl), and that Ab4 share idiotypic determinants with Ab2. Moreover, this finding of idiotypic similarity between Abl and Ab3 on one hand, and Ab2, Ab4 on the other hand, led the immunologists to the concept of a circular idiotypic network 14. Anti-idiotypic antibodies have also been reported to occur naturally in the sera of parous women 15. It has been shown that the sera of parous women contain anti-idiotypic antibodies directed against regulatory idiotypes of anti-hla DR antibodies (in parous women anti-hla antibodies are found that are directed against some HLA antigen inherited by the fetus from the father). Such antibodies have been postulated to play a key role in downregulating the maternal reactions against the fetus 15. Furthermore, in renal allograft recipients 16, the presence of anti-

12 idiotypic antibodies to HLA were shown to be positively correlated with the success in transplantation, indicating their potential immunosuppressive effect 16. The production of auto-anti-idiotypic antibodies and auto-antiidiotypic lymphocytes has been shown to occur in many systems that use conventional antigens such as haptens, proteins, and viruses 17. One of the earliest illustrations of this phenomena was studied by Rodky 18 in which he immunized outbred New Zealand white rabbits with p-aminophenyl N-trimethylammonium chloride (TMA)-KLH conjugate and collected the sera for 189 days. The anti-tma antibodies were purified by affinity chromatography, then the same animal that synthesized the antibody was reinjected with polymerized F(ab)2 of anti-hapten antibodies. The sera were collected after autoimmunization. The results showed that these rabbits made an anti-idiotypic auto-antibody and the reaction between the anti-idiotypic auto-antibody and anti-tma antibody could be inhibited with the hapten. Moreover, each rabbit in this study responded to anti-tma antibodies produced by itself, and not the anti-tma made in other individuals 18. This experiment clearly demonstrates that one individual is able to synthesize antibodies specific for its own V regions. The immune response to foreign lymphocytes may also include the production of auto-anti-idiotypic antibodies. The immune response of the immunocompetent host to foreign lymphocytes major histocompatibility complex antigens (anti-foreign MHC) is well known. However, the injected immunocompetent lymphocytes may respond (transiently) to the host alloantigen and produce a graft-

13 versus-host (GVH) reactivities. The host, in turn, might produce antibodies against the immunogenic donor's T cell receptor which is capable of recognizing the host. This anti-idiotypic response is an "anti-anti-self' response 12 and has been observed by Ramseier 19, Ramseier and lindenmann 20, Binz and Wigzell 21 ' 22-23, and Hoffmann et. al. 12. In the Ramseier and Lindenmann study 20, immunization of (A x B)F1 animals (rats, mice, and Syrian hamster) with lymphoid cells from parent A could induce anti-idiotypic antibodies to the alloantigen receptors on the parental lymphocytes in all three species. Such anti-idiotypic antibodies prevented the recognition of parental strain A against the Fl lymphocytes in vitro (Ramseier and Lindenmann used the term "anti-recognition structure" instead of anti-anti-self). In Binz and Wigzell studies 21, T lymphocytes from strain A animal treated with Fl anti-(a anti-b) (this is anti-anti-b) antibodies and complement failed to react to strain B alloantigens, but reacted to a third party alloantigen in GVH assay. Moreover, anti-(a anti-b) antibodies produced in F1(A x B) against the A parental receptor that recognize the B alloantigen were able to agglutinate SRBC coated with A anti-b antibodies 21. The results indicated that anti-anti-self antibodies produced in Fl (injected with parental T lymphocytes) recognized idiotypic determinants shared both by B cells and T lymphocytes 21. However, the Binz and Wigzell work was controversial, since despite concerted efforts by Bellgrau and Wilson 24» anti-idiotypic antiserum to the anti-mhc T cell receptors could not be observed.

14 Hoffmann et al. 12 further investigated the studies of Ramseier and Lindenmann, and Binz and Wigzell with a different assay using alloantisera from congenic or fully allogeneic strains of mice. Based on the results of their experiments, a new symmetrical relationship emerged from considering the interactions that occurs when lymphocytes from two different immune system networks confront each other. The relationship between the reciprocal responses of two immune system network was called "second symmetry" by Hoffmann 12. For example, when mice of strain A are immunized with fully allogeneic lymphocytes from mice of strain B, the A cells that recognize the B host antigens are stimulated and could transiently proliferate, whereas the cells of other specificities are not stimulated. Two sets of foreign entities that are recognized by B are then, A MHC antigens, and the A anti-b MHC receptors. Thus, the immune response of B to A are B anti-a (conventional anti-foreign) and B anti-(a anti-b) which is anti-anti-self. Likewise, the immune response of an A animal to B lymphoid cells are A anti-b (antiforeign) and A anti-(b anti-a) which is anti-anti-self. Moreover, the anti-foreign response of strain A (that is A anti-b) is complementary (has V region that fit) to the anti-anti-self response of strain B [that is B anti-(a anti-b)], and vice versa (Figure 1). Therefore, the two components in responses of A to B are complementary to the two components in the response of B to A. This relationship between the two reciprocal responses of two immune system network was called second symmetry by Hoffmann 12. Second symmetry is supported by several pieces of experimental evidence. Anti-anti-self antibodies were specifically

15 A anti-b receptor A MHC B enti-a receptor eptor/- "v. 7 B 1 B MHC\ D J A's response to B: A enti-b MHC A enti-(b anti-a receptor) B's response to A: B anti-a MHC B anti-(a anti-b receptor) Figure 1. The Immunization of mice with allogeneic lymphocytes produces anti-foreign and anti-anti-self MHC responses. An immune response in an animal of strain A to allogeneic cells from strain B consists of two components : the conventional anti-foreign (A anti-b) response, and the response of A to the receptors on B strain cells that recognize A (that is, "A anti-(b anti-a)', which is anti-anti-a MHC). The converse immunization results in the production of B anti-a and B anti-(a anti-b) responses. The total (anti-foreign plus anti-anti-self) in the A anti-b response is complementary (has V regions that fit) to the total B anti-a response. Adapted from Hoffmann, G. W., Cooper-Willis, A., and Chow, M., J. Immunol. 137, 61-68, 1986 (ref. 12).

16 able to inhibit the cytotoxicity of alloantibody. For example, A anti-(b anti-a) antibodies (that is A anti-b antiserum produced in A mice injected by B lymphocytes) inhibited the cytotoxicity of B anti-a antibodies against strain A target cells 12. Anti-anti-self antibodies are interpreted to have "self MHC image" specificity, in that they are able to specifically inhibit the cytotoxicity of polyclonal anti-mhc antibodies in reciprocal alloantiserum 12. Furthermore, anti-anti-self antibodies were able to enhance the survival of skin allografts. Strain A mice injected with B anti-(a anti-b) antibodies specifically retained B skin grafts longer than third party skin allografts 12. This phenomena has been called "reverse enhancement" because the alloantiserum used to treat the recipient is made in the donor, in contrast to conventional enhancement which uses anti-graft antiserum made in the recipient 25. In a separate experiment a direct avidin-biotin ELISA was developed and tested for the detection of anti-anti-self antibodies 26. It was demonstrated in this assay that biotinylated A anti-b antiserum binds only to the reciprocal B anti-a antiserum, and not to normal or itself 26. In this project Hoffmann's second symmetry model was tested by raising reciprocal alloantisera in different strains of alloimmune mice. The complementarity of anti-foreign and anti-anti-self antibodies between two reciprocal sets of alloantisera was investigated by using alloantisera and affinity purified serum IgG in an avidin-biotin ELISA. The specificity of anti-anti-self antibodies in B6 anti-balb/c was investigated by the ELISA, and the speculated self MHC image characteristics of anti-anti-self antibodies produced

17 9 in alloimmune B6 anti-balb/c and old B6 was shown by the inhibition of antibody and complement mediated cytotoxicity.

18 Materials and Methods Mice : Mice of the inbred strains Balb/cJ (Balb/c, H-2 d ), C57BL/6J (B6, H-2*>) and CBA/J (CBA, H-2^) were obtained from Jackson Laboratories (Bar Harbour, ME) or raised in our breeding colony from stocks obtained from the Jackson. Mice used for immunizations were female 6-8 weeks old at the beginning of the immunizations. Preparation of Lymphoid Cell Suspensions : Spleen, thymus, and lymph nodes (cervical, mesenteric, inguinal, and auxiliary) were aseptically removed and single cell suspensions were prepared by pressing through a stainless steel mesh in phosphate buffered saline (PBS). The cells were washed three times with PBS by centrifugation before being resuspended at appropriate concentration for injection. The cell number was determined with a hemocytometer and the cell viability was determined by 0.4% w/v trypan blue dye exclusion and was always greater than 80%. Alloantisera. Seven mice in each group were immunized with 10 weekly interperitoneal (i.p.) injections of 5 x 10 7 viable allogeneic lymphoid cells (lymph node, spleen and thymus) in 0.2 ml phosphate buffered saline, ph 7.2 (PBS) and bled 7 days after the last injection. Thereafter, mice were injected with 1 x 10 7 allogeneic lymphoid cells (i.p.) 7 days prior to bleeding. Mice were bled by tail bleeding under anesthesia with halothane. The blood was clotted for

19 11 1 h at 37 C and refrigerated overnight at 4 C to allow for clot retraction before being processed by centrifugation for the sera. The antisera were heat inactivated in a waterbath at 56 C for 30 minutes and stored at -20 C in a freezer until use. Reciprocal sets of antisera were raised between the following pairs of mice: Balb/c and B6, Balb/c and CBA, and B6 and CBA. The same number of matched controls were injected with the same dose of syngeneic lymphoid cells in case of B6 and Balb/c or injected with PBS, unless otherwise stated. (Note: Alloimmune Balb/c and B6 received 5 X 10 7 each of the other strain's lymphoid cells emulsified 1 to 1 (v/v) ratio in Complete Freund's Adjuvant after 10 weeks of immunization and prior to antibody dependent and complement mediated cytotoxicity experiments). Purification oflgg from the sera : The sera were diluted 1 to 1 (v/v) in 0.5 M NaCl-PBS ph 7.3 and loaded onto the Goat anti-mouse IgG immunoabsorbant columns (Sigma). The antibodies (IgG) were eluted with 0.15 M NaCl, 0.1 M HCl-glycine, ph 2.3, and immediately neutralized with 1.0 M TRIS base, ph 8.0. All concentrating was done with Centriprep-30 (Amicon Corporation, Lexington, MA) cartridge according to the manufacturer's protocol, followed by dialysis in PBS at4 C Biotinylation of antibodies. Purified antibodies in PBS 27 (2 mg/ml) or antisera 28 diluted 1 in 10 in PBS were biotinylated by adding 0.1 volume of biotinylation reagent [5 mm biotinamidocaproyl N-hydroxysuccinimide ester (Sigma) in dimethyl-

20 formamide] and incubating at room temperature for 4 hours with occasional mixing. The dimethyl-formamide and unused biotinylation reagent were removed by dialysis against several changes in PBS at 4 C in the cold room. Protein determination: The concentration of IgG was estimated by measuring U.V. absorbance at 280 nm on Pharmacia GeneQuant spectrophotometer. It was assumed that 1 A 280 nm= 0.69 mg/ml mouse gamma globulin (IgG) 29. Sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) : The samples were boiled for 5 minutes at 100 C boiling water in nonreducing sample buffer and subjected to 7.5% SDS-PAGE using the discontinuous conditions and buffer system of Laemmli 30. The electrophoresis was carried out using a BIO-RAD (Bio-Rad, Mississauga, Ontario) minigel apparatus under constant voltage (100V) for 1 hour. Gels were stained with Coomassie Brilliant blue R- 250, then de-stained with a solution of 10% glacial acetic acid and 25% methanol. Second symmetry ELISA : The sera or the affinity purified sera IgG were coated (50 ^il/well) directly to Immulon 2 ELISA plates (Dynatech Laboratories, INC) in 0.05 M Carbonate-Bicarbonate buffer ph 9.6 (Sigma Chemical Co. St. Louis, MO) at 4 C overnight. All washes were done with PBS containing 0.05% Tween20 (PBST20). The plates were blocked with 5% fat-free casein (BDH Chemical Ltd., Poole, England) in PBST20 for 1 hour at 37 C. Then, the plates were

21 (500 ng/well) in PBST20 with 1% casein for 3 hours at 37 C. The plates were incubated for another hour with strept. avidin-alkaline phosphatase conjugate (Calbiochem) diluted 1/2000 in PBST20 with 1% casein and the bound antibodies were detected with p- nitrophenyl phosphate, Disodium substrate (Sigma) dissolved in 10% diethanolamine 26. The absorbances at 405 nm were read after 20 to 45 minutes on the Bio-Rad model 450 microplate reader. All incubations were done at 37 C, and all wells were done in duplicate, except for the individual mice sera (Figure 2c) wells were done in triplicates. Measurement of alloantibody by 51 Cr release assay : The assay used for these experiments is the same as the one previously described!2. The medium used for the assay is RPMI 1640 (Gibco Laboratories, Grand Island, NY) supplemented with HEPES buffer (Gibco, 25mM), 0.37% sodium bicarbonate and 15% fetal bovine serum (Gibco). A single cell suspension of lymph node cells was prepared in medium and labeled for 90 minutes at 37 C in Na2 51 Cr04 (ICN Biomedical Canada. Ltd. St. Laurent, Quebec), washed twice in the same medium and resuspended to 2 x 10^ cells/ml. Serum was diluted in medium and further doubling dilutions were prepared in round bottom microtitre trays (Linbro, Mclean, VA) in a final volume of 50 nl. 50 fil of the cell suspension was added and the plates were incubated at 37 C for 30 minutes. 100 pd of medium was added to each well, the plates were centrifuged at 500 X g (Beckman CS-6R) for 5 minutes and the medium was flicked off. 25 fxl of diluted (1/20 in medium) Low Tox-M rabbit complement

22 (Cedarlane-previously absorbed with 1 x 10& mouse spleen cells per ml of complement on ice for 1 hour and stored at -20 C until use) was added to each well and the plates were gently vortexed, then incubated for 60 minutes at 37 C. 175 jul of medium was added to each well, the plates were centrifuged at 500 X g for 5 minutes and 100 jui of supernatant was removed for gamma counting in a Picker Pace-1 gamma counter. Controls included incubating the cells in the presence of complement only. Maximum release was determined by incubating the cells during the 2nd step in 25 ul of 5% Triton X-100 in HEPES-buffered salt solution (HBSS). All samples were run in triplicate. Results are expressed as a percentage of specific lysis resulting from the formula: % lysis = [(observed cpm - C cpm) / (total cpm - C cpm)] X 100 where observed cpm is target cells incubated with alloimmune or normal sera, C cpm is target cells incubated in complement alone, and total cpm is from target cells solublized with detergent (5% Triton X-100 in HBSS). Measurement of anti-anti-self (MHC image) antibodies by the inhibition of lytic antibodies detected with 5lcr release : Cells were prepared as described for the above assay. Sera (inhibitory or normal) is diluted in medium to a volume of 25 \d and 25 ul of diluted "lytic" antibody is added and incubated for 30 minutes at 37 C. The cells are added to the plates and the assay is developed as previously described 10. An additional control is the presence of cells

23 and lytic antibody in the absence of any inhibitors. Percent inhibition is calculated as: observed cpm - C cpm % Inhibition = [l - ( ) ] X 100 lytic Ab cpm - C cpm Where observed cpm= cpm for lytic alloantisera in the presence of inhibitory or normal serum. C cpm= cpm for target cells in the presence of complement alone (no serum) lytic Ab cpm= cpm for lytic alloantiserum in the absence of any inhibitors.

24 Results Anti-anti-self MHC antibodies detected by the second symmetry ELISA. Anti-anti-self antibodies in alloimmune serum : Balb/c anti-b6 antiserum raised by repeated immunization of Balb/c mice with B6 lymphocytes is expected to contain two classes of specific antibodies, namely, Balb/c anti-b6 (anti-foreign) and Balb/c anti-(b6 anti- Balb/c) which is anti-anti-balb/c. Similarly, the two specific classes of antibodies expected to be found in B6 anti-balb/c are B6 anti- Balb/c and B6 anti-( Balb/c anti-b6), the latter being anti-anti-b6 antibodies. Moreover, the anti-anti-balb/c (anti-anti-self) antibodies present in the Balb/c anti-b6 are considered to be complementary to anti-balb/c (anti-foreign) antibodies in the B6 anti-balb/c antiserum, and vice versa (as shown in Figure 1, page 6). The second symmetry model was tested using the second symmetry ELISA. In this assay alloimmune(i.e A anti-b) or normal sera were used to coat the ELISA plates and were considered to be analogous to the inhibitory sera in the inhibition of cytotoxicity assay. Biotinylated reciprocal alloantisera (i.e. B anti-a, analogous to the lytic serum in the inhibition of cytotoxicity assay) was then allowed to react with the antibodies bound to the plate, and the bound biotinylated antibodies were detected with avidin-alkaline phosphatase conjugate and the appropriate substrate 26. The binding of one set of alloantisera to the

25 biotinylated antisera was considered to be mainly due to interactions of anti-anti-self antibodies to anti-foreign in it's reciprocal. Binding of Biotinylated Balb/c anti-b6 to B6 anti-balb/c. sera : After 10 weeks of alloimmunization a significant level of binding occurred between biotinylated Balb/c anti-b6 and B6 anti-balb/c antisera on the ELISA plate. As shown in Figure 2a, biotinylated Balb/c anti-b6 antiserum binds to the reciprocal, B6 anti-balb/c, but does not bind significantly to age matched B6 PBS injected mice sera. Additional controls used are Balb/c anti-b6 and Balb/c PBS injected mice sera. This resulting signal is interpreted as binding of biotinylated anti-b6 MHC antibodies in Balb/c anti-b6 antiserum to the anti-anti-b6 antibodies in the reciprocal, B6 anti-balb/c antiserum on the plate or vice versa (since the relationship between Balb/c anti-b6 and B6 anti-balb/c is believed to be "symmetrical"). The binding between the two different sets of alloantisera is believed to occur due to V region complementarity between anti-foreign MHC in one set of alloantisera and anti-anti-self (self MHC image) in the reciprocal alloantisera.

26 Dilution of serum used to coat ELISA plate Figure 2a. Binding of biotinylated Balb/c anti-b6 antiserum to the B6 anti-balb/c detected by the second symmetry ELISA. In this assay, the ELISA plates were directly coated with the alloimmune or PBS injected mice sera at the indicated dilutions. After incubation with biotinylated Balb/c anti-b6 alloantiserum, the bound antibodies were detected with Strept. avidin-alkaline phosphatase conjugate and substrate. As shown, biotinylated Balb/c anti-b6 binds to B6 anti-balb/c ( ), but does not bind to B6 PBS injected mice serum(#). Additional controls are Balb/c anti-b6 ( ) and B6 PBS injected serum(o). Biotinylated Balb/c anti-b6 antiserum was diluted to a final concentration of 1/500. Each point represents the mean and standard deviation (SD) of duplicates. Where no SD is shown, the SD was smaller than the symbol used.

27 Binding of biotinylated Balb/c anti-b6 to old B6 mice sera: During the course of this project, it was found that biotinylated Balb/c anti-b6 antiserum not only binds the reciprocal, B6 anti- Balb/c (Figure 2a), but also binds to old B6 mice sera. As shown in Figure 2b, biotinylated Balb/c anti-b6 antiserum binds to 1 year, and 1.5 year old B6 PBS injected mice serum, but does not bind to the age matched (17 weeks old) B6 PBS. The result is interpreted as binding of anti-b6 MHC antibodies in Balb/c anti-b6 to anti-anti-b6 antibodies in old B6 sera coated on the ELISA plate. Moreover, a higher level of binding is observed between biotinylated Balb/c anti- B6 to 1.5 year old B6 PBS than 1 year old B6 PBS, so production of presumed anti-anti-b6 (B6 MHC image) antibodies in B6 mice is correlated with aging (Figure 2b).

28 Figure 2b Dilution of serum used to coat ELISA plate Figure 2b. Binding of biotinylated Balb/c anti-b6 to PBS injected 1 year and 1.5 year old mice serum detected by the second symmetry ELISA. Biotinylated Balb/c anti-b6 binds not only to the reciprocal B6 anti- Balb/c serum(b), but also to 1 year (A) and 1.5 year old B6 injected with PBS ( ). Additional controls are serum prepared from 1 year old Balb/c PBS (A) and 17 weeks old B6 PBS ( ). This resulting signal is interpreted as binding of biotinylated anti-b6 MHC antibodies in Balb/c anti-b6 antiserum to the anti-anti-b6 (B6 MHC-image) antibodies in the 1 year and 1.5 year old B6 PBS injected mice serum on the plate. Each point represents the mean and standard deviation of duplicates.

29 Binding of biotin.yla.ted Balb/c anti-b6 to individual B6 anti- Balb/c and 1.5 year old B6 mice sera: In the previous results (Figure 2 a, and Figure 2 b), pooled serum from 7 mice in each group was used to detect anti-anti-self antibodies. In order to determine the level of anti-anti-self antibodies produced in each of the alloimmune and 1.5 year old B6 mice, individual serum was used in the second symmetry ELISA. As shown in Figure 2c, high level of binding is observed between the biotinylated Balb/c anti-b6 and all of B6 anti-balb/c individual sera on the plate. A high level of binding is also obtained between the binding of Balb/c anti-b6 and six out of seven individual 1.5 year old B6 PBS injected mice serum with one binding moderately. In this assay, none of the seven 17 weeks old B6 PBS injected mice serum bound significantly to the biotinylated Balb/c anti-b6 (Figure 2c). Therefore, the presumptive anti-anti-self antibodies are present in all of alloimmune B6 anti-balb/c and 1.5 year old B6 PBS injected mice serum.

30 1.4 Figure 2c 17 weeks old B6 PBS B6 anti-balb/c 1.5 YR. B6 PBS 17 weeks old B6PBS B6 anti-balb/c 1.5 Year old B6 PBS Figure 2c. Binding of biotinylated Balb/c anti-b6 to individual B6 anti-balb/c and 1.5 year old B6 PBS injected mice serum in the second symmetry ELISA. Bioinylated Balb/c anti-b6 pooled serum binds to the individual B6 anti-balb/c ( ), and 1.5 year old B6 PBS ( ) injected mice serum on the plate, but does not significantly bind to 17 weeks old B6 PBS injected mice serum ( ). The final dilution of each coated serum was 1/1000 and the final concentration of the biotinylated Balb/c anti-b6 was 1/500. Each point represents the mean and standard deviation of triplicates.

31 Biotinylated Balb/c anti-b6 binds to its reciprocal B6 anti- Balb/c and not to other alloantisera or syngeneically immunized mice sera : It was of particular interest to see whether there is cross reactive binding of biotinylated Balb/c anti-b6 alloantiserum to any other raised alloantiserum on the ELISA plate. Additional controls of B6 and Balb/c syngeneically immunized mice sera were also included. As can be seen in Figure 2d, biotinylated Balb/c anti-b6 binds significantly to the reciprocal B6 anti-balb/c on the plate, but does not bind to any other alloantiserum on the plate. The complementarity of anti-mhc and anti-anti-self antibodies in two sets of alloantisera namely Balb/c anti-b6 and B6 anti-balb/c is thus highly specific. Moreover, the biotinylated Balb/c anti-b6 does not react with the syngeneically immunized B6 mice sera (Figure 2d). This is because the sera of syngeneically immunized B6 lacks the "anti-anti-b6" antibodies.

32 Figure 2d < sa Dilution of serum used to coat ELISA plate Figure 2d. Binding of biotinylated Balb/c anti-b6 to the reciprocal B6 anti-balb/c, but not to any other alloantiserum or syngeneically immunized mice sera. Biotinylated Balb/c anti-b6 binds specifically to it's reciprocal B6 anti-balb/c ( ) on the plate. Balb/c anti-b6 does not bind to other alloantisera namely Balb/c anti-cba (m), CBA anti-balb/c (0), CBA anti-b6 (4), and B6 anti-cba ( ). Biotinylated Balb/c anti-b6 does not bind to B6 syngeneic ( ) and 17 weeks old B6 PBS ( ). An additional control is serum from Balb/c syngeneically (A) immunized mice. Each point shows the mean and standard deviation of duplicates.

33 Binding of biotinylated B6 anti-balb/c to its reciprocal Balb/c anti-b6 sera : In the next stage of this project, the reciprocal direction of binding observed in Figure 2a was determined by second symmetry ELISA. Figure 3 shows the binding between the biotinylated B6 anti-balb/c and it's reciprocal Balb/c anti-b6. The resulting signal is interpreted as binding of biotinylated mainly, anti- Balb/c MHC antibodies in B6 anti-balb/c antiserum to the anti-anti- Balb/c (Balb/c MHC-image) antibodies in the reciprocal, Balb/c anti- B6 antiserum on the plate and vice versa. Comparing the level of binding of biotinylated B6 anti-balb/c to the reciprocal Balb/c anti- B6 (Figure 3) to that of binding of biotinylated Balb/c anti-b6 to B6 anti-balb/c antiserum alloantiserum (Figure 2a), we find that there is higher level of binding in the latter. Since the second symmetry ELISA is not conclusive in determining which set of the alloantiserum (Balb/c anti-b6 or B6 anti-balb/c) contains more anti-anti-self antibodies, additional approaches are taken that will be presented hereinafter in this section.

34 4) c * 1 o «C Figure 3 Balb/c anti-b6 Balb/c PBS B6 anti-balb/c B6PBS.S u = 0.1- C O I rt Dilution of serum used to coat ELISA plate Figure 3. Binding of biotinylated B6 anti-balb/c to Balb/c anti-b6 in second symmetry ELISA. Biotinylated B6 anti-balb/c binds to it's reciprocal, Balb/c anti-b6 ( ). It does not bind Balb/c PBS (O) or B6 PBS ( ). An additional control is B6 anti-balb/c ( ). The resulting signal is interpreted as binding of mainly, anti-balb/c antibodies in B6 anti-balb/c antiserum to the anti-anti-balb/c antibodies in the reciprocal, Balb/c anti-b6 on the plate and vice versa. Each point represents the mean and standard deviation of duplicates.

35 Anti-anti-self antibodies in alloimmune Balb/c and B6 and old B6 mice, using affinity purified IgG : In the next stage of this project it was decided to establish whether the binding of one set of alloantiserum to the reciprocal observed in the second symmetry ELISA is mainly due to the antibodies. By using immunoglobulin rather than antisera the possibility of significant binding between the non-immunoglobulin components present in antisera on the ELISA plates and biotinylated detecting reagents can be ruled out. Therefore, goat anti-mouse IgG immunoabsorbant columns were used to purify the IgG fraction of the sera and the purified IgG was used for coating the plates and as biotinylated detecting reagent in second symmetry ELISA. As shown in figure 4 and figure 5 a pattern of binding is observed that is similar to that seen in figure 2 b and figure 3 respectively for sera. Therefore, it can be concluded that this binding is mainly due to IgG. Additional controls were the use of the sera that were highly depleted of the immunoglobulin fraction by 50% saturated ammonium sulfate (SAS) and then run the supernatant over the protein A affinity column several times before being used for the second symmetry ELISA. Such treatment abolished the binding capability of one set of alloantiserum to the reciprocal alloantiserum completely (data not shown). The purity of the purified IgG was verified by the nonreducing SDS-PAGE (Figure 6). A single IgG band was observed after staining with the Coomassie blue indicating high purity of the eluents.

36 Figure 4 and Figure 5. anti-anti-self antibodies in alloimmune sera detected by the second symmetry ELISA. Alloimmune or PBS injected sera IgG were directly coated at the indicated concentration. After incubation with biotinylated alloantisera IgG, the bound antibodies were detected with Strep, avidin-alkaline phosphatase conjugate and substrate. The resulting signal is interpreted as resulting from the binding of biotinylated anti-foreign MHC antibodies to the anti-anti-self (self MHC-image) antibodies on the plate. Each point represents the mean and standard deviation of duplicates. Figure 4. Biotinylated Balb/c anti-b6 alloantiserum IgG binds to B6 anti-balb/c antiserum IgG ( ), and 1 year old B6 mice injected with PBS (A), and 1.5 year old B6 mice injected with PBS ( ), but neither 17 weeks old B6 mice injected with PBS ( ) nor 17 weeks Balb/c mice injected with PBS (O). Additional control is Balb/c anti-b6 ( ). Figure 5. Biotinylated B6 anti-balb/c alloantiserum IgG binds to it's reciprocal, Balb/c anti-b6 ( ). B6 anti-balb/c does not bind to Balb/c (O) or B6 mice injected with PBS ( ) serum IgG. Additional control is B6 anti-balb/c ( ). The final concentration of the biotinylated IgG used was 500 ng/well. Each point represents the mean and standard deviation of duplicates.

37 29 Figure 4 o.5 eo T3 - SvD XI CO 0.8 a c o c CO Figure 5 2 o Balb/c anti-b6 Balb/c PBS B6 anti-balb/c B6PBS.5 u ^ -"rt 0.1 B CO C ' * *. 1 * 4-1 o rt < CO I I I ' I ' I ' I ' I I I ng of IgG used to coat ELISA plate

38 7.5% Non-reducing SDS-PAGE Figure 6. Purity of the affinity purified mouse sera IgG established by non-reducing SDS-PAGE. Samples of protein were separated by electrophoresis on a 7.5% nonreducing SDS polyacrylamide gel and stained with Coomassie Brilliant blue. Lane 1 is purified rabbit IgG used as molecular weight standard (approximately 3 ng.). Lane 2 is 1.5 il of a sample of whole mice serum. Lane 3 Balb/c anti-b6 IgG. Lane 4 Balb/c PBS IgG. Lane 5 B6 anti-balb/c. Lane 6 B6 PBS. Lane 7 is a sample of runthrough depleted of IgG. Approximately 3 ng. of each sample was used.

39 Anti-foreign MHC antibodies induced in alloimmune Balb/c and B6 detected using antibody and complement mediated cytotoxicity assay. Anti foreign MHC antibodies in alloimmune Balb/c and 56 : As stated earlier, the binding of the biotinylated Balb/c anti-b6 to the reciprocal B6 anti-balb/c on the plate was considerably higher than the binding of biotinylated B6 anti-balb/c to the Balb/c anti-b6 on the plate. Initially it was believed that this could be due to a higher level of anti-mhc (anti-foreign) antibodies in one set of alloantiserum than the other. To enhance the immune response, mice that had been immunized with allogeneic cells were boosted with the same cells emulsified 1:1 (v/v) in complete Freund's adjuvant (CFA) given subcutanteously. The second symmetry ELISA was then repeated. However, no significant increase in the level of binding was observed in either direction (data not shown). The second symmetry ELISA can give a weak signal due either to the absence or low level of anti-foreign (anti-mhc) antibodies or of anti-anti-self (MHCimage) antibodies. Therefore, in order to further assess the level of anti-foreign (anti H-2 d and anti H-2 b respectively) antibodies in Balb/c anti-b6 and B6 anti-balb/c, antibody and complement mediated cytotoxicity was performed (Figure 7 and Figure 8). As can be observed in Figure 7, Balb/c anti-b6 were producing highly cytotoxic antibodies. At 1/50 dilution, Balb/c anti-b6 killed 86%, while Balb/c normal sera killed only 5% of the B6 target cells. Similar results were obtained for B6 anti-balb/c alloantiserum. In

40 this assay B6 anti-balb/c killed 90% of Balb/c target cells, whereas, B6 normal serum killed 0% (Figure 8). These results (Figure 7 and Figure 8) showed that both Balb/c anti-b6 and B6 anti-balb/c were highly and almost equally producing anti-foreign antibodies.

41 Cytotoxicity of Balb/c anti-b6 (Figure 7), and B6 anti-balb/c (Figure 8) alloantisera on B6 and Balb/c lymph node cell targets respectively. The direct cytotoxicity assay detects interactions between the MHC antigens on the target cells and the anti-mhc antibodies in the alloimmune serum. 51 Cr-labelled lymph node cells were incubated with the antisera for 30 minutes, washed and incubated with complement for 60 minutes. The amount of 51 Cr released into the supernatant is proportional to the amount of antibody plus complement mediated killing. Figure 7. Cytotoxicity of Balb/c anti-b6 ( ) alloantiserum, and Balb/c normal serum (O) on B6 target cells. Figure 8. shows the lysis of Balb/c lymph node targets by B6 anti- Balb/c ( ) and B6 normal sera ( ). Each point represents the mean and standard deviation of triplicates.

42 34 v 60 u n CQ o Figure 7 Balb/c anti-b6 Balb/c Normal u ft * Dilution of sera used a) 00 l-c Figure 8 B6 anti-balb/c B6 Normal rt pa o 'vi \ 60- SO' Dilution of sera used

43 Anti-anti-self antibodies in alloimmune and old B6 detected by the inhibition of antibody and complement mediated cytotoxicity. The presence of anti-anti-self MHC in alloimmune Balb/c and B6 and old B6 investigated by the inhibition of antibody and complement mediated cytotoxicity: In order to establish which set of allogeneic mice were producing more anti-anti-self MHC antibodies, inhibition of alloantibody and complement mediated cytotoxicity was performed (since the second symmetry ELISA assay is not conclusive in determining whether Balb/c anti-b6 or B6 anti-balb/c is producing more anti-anti-self antibodies). The inhibition of cytotoxicity assay detects anti-anti-self (MHC-image) antibodies in one alloimmune serum by the inhibition of cytotoxicity of the reciprocal alloimmune serum against its target cells. As shown in Figure 9, there was no significant difference between inhibition by Balb/c anti-b6 and Balb/c normal serum and neither is capable of inhibiting the cytotoxicity of B6 anti-balb/c against Balb/c target cells. This assay failed to show any significant presence of anti-anti-self (Balb/c MHC-image) antibodies in Balb/c anti-b6. However, the same assay resulted in inhibition of killing of B6 target cells by Balb/c anti-b6 alloantiserum using B6 anti-balb/c and serum from 1.5 year old B6 PBS injected mice, but not with normal (10-12 weeks old) B6 serum (Figure 10). The inhibition occurred at 1/10 dilution of inhibitory B6 anti-balb/c and 1.5 year old B6 were 50% and 38% respectively, whereas weeks old B6 serum caused only 14% inhibition. The inhibitory effect of B6 anti-

44 Balb/c and 1.5 year old B6 injected with PBS is interpreted to be due to the presence of anti-anti-b6 (B6 MHC-image) antibodies in these sera which bind to anti-b6 MHC in the Balb/c anti-b6 alloantiserum and prevent it from killing B6 target cells. Moreover, these findings in figure 10 showing the presence of anti-anti-self antibodies in alloimmune and 1.5 year old B6 are consistent with the findings of the second symmetry ELISA in figure 2b.

45 Figure 9. Absence of anti-anti-self antibodies in Balb/c anti-b6 alloantisera as detected by the inhibition of cytotoxicity. 51 Cr-labelled B6 lymph node cells were incubated with the inhibitory (Balb/c anti-b6) and the lytic B6 anti-balb/c antisera for 30 minutes, washed and incubated with the complement for 60 minutes. Cell lysis was determined by measuring the amount of 51 Cr released into the supernatant. Inhibition of killing of Balb/c lymph node target cells by B6 anti-balb/c lytic antiserum using Balb/c anti-b6 ( ) and normal (10-12 weeks old) Balb/c (O) serum. B6 anti-balb/c antiserum when diluted 1/50 in the media plus complement killed 81% of Balb/c target cells in the absence of any inhibitors in this instance. Each point represents the mean and standard deviation of triplicates. Statistically (by unpaired Student t test at 1/10 dilution of inhibitors) there is no significant difference between inhibition by Balb/c anti-b6 and Balb/c normal serum and neither is capable of inhibiting the cytotoxicity of B6 anti-balb/c against Balb/c target cells.

46 38 60 Figure 9 Balb/c anti-b6 Balb/c Normal Dilution of inhibiting serum

47 Figure 10. Anti-anti-B6 (B6 MHC-image) antibodies in the serum of B6 anti-balb/c and 1.5 year old B6 PBS injected mice serum. Inhibition of killing of B6 target cells by Balb/c anti-b6 using B6 anti-balb/c alloantiserum ( ), and serum from 1.5 year old B6 PBS injected mice (A), but not with normal (10-12 weeks old) B6 ( ) serum. 1/50 diluted Balb/c anti-b6 antiserum in medium plus complement killed 67% of B6 target cells in the absence of any inhibitors. Each point represents the mean and standard deviation of triplicates. Statistical analysis of the data (at 1/10 dilution) by unpaired Student t test shows that B6 anti-balb/c and 1.5 year old B6 mice sera are different from the serum of normal (10-12 weeks old) B6 with P value of and respectively.

48 % specific inhibition of lysis of B6 target cells O c o s 9 cr s CtQ VI "1 e 3 CO OS 7, o i-* <yi < P CO Us n ON Co 3 CO p CT O 4^ O

49 In addition to raising the reciprocal alloantisera between Balb/c and B6, alloimmunization also was done among the following group pairs : Balb/c and CBA (producing Balb/c anti-cba, and separately CBA anti-balb/c), and B6 and CBA (producing B6 anti-cba and CBA anti-b6). After alloimmunization for 10 weeks, the sera were tested for the presence of anti-anti-self antibodies using the second symmetry ELISA, but no significant difference between the alloitnmune and normal sera were observed in this assay (data not shown) even though alloimmunization between all of the above strain combinations were shown to produce alloantibodies (data not shown). This is in contrast to the results of Dr. Cooper-Willis 12, who observed the presence of anti-anti-self antibodies using the B6 and CBA strain combinations as well as 5 other mice strain combinations by the inhibition of antibody and complement mediated cytotoxicity. The reason for this discrepancy is unknown.

50 Discussion It is known that T lymphocytes express reactivity toward allo- MHC structures as examplified by strong proliferation in primary MLC and efficient induction of GVH reactions 3!»32. in this project, the presence of antibodies produced by the host against such allo-mhc TCR was investigated indirectly via the second symmetry model 12. The relationship between anti-foreign and anti-anti-self antibodies was investigated by a second symmetry ELISA and inhibition of antibody and complement mediated cytotoxicity. After 10 weeks of alloimmunization with lymphoid cells, a significant level of binding between biotinylated Balb/c anti-b6 and it's reciprocal, B6 anti- Balb/c antiserum was observed in the second symmetry ELISA (Figure 2a.). This specific binding was attributed to complementarity of anti-anti-self and anti-foreign MHC antibodies in alloimmune Balb/c and B6 mice. However, during the course of this project it was found that in addition to binding B6 anti-balb/c antiserum, biotinylated Balb/c anti-b6 also binds to 1 year and 1.5 year old B6 mice that have not received any alloimmunization. This resulting signal was interpreted mainly as binding of biotinylated anti-b6 MHC antibodies in Balb/c anti-b6 to the anti-anti-b6 (B6 MHC-image) antibodies in the 1 and 1.5 year old B6 PBS injected mice sera on the plate. As shown (Figure 2b) there was a higher level of binding of biotinylated Balb/c anti-b6 to 1.5 year old B6 PBS than 1 year old B6 serum, so production of presumptive anti-anti-b6 (B6 MHC-image) antibodies in B6 mice is likely to be correlated with advances in

51 aging. Results shown by the inhibition of antibody and complement mediated cytotoxicity (Figure 10) were consistent with the finding of the second symmetry ELISA (Figure 2b). Antibodies that are images of self MHC may be part of the normal repertoire as previously suggested by Rossi et al 33. In their experiments, some culture supernatants from Balb/c LPS-stimulated splenocytes were shown to react to anti I-A d and anti I-E d coated on the ELISA plate 33. The presence of anti-anti-self MHC antibodies in old B6 mice might suggest a set of mechanisms participating in the maintenance of natural tolerance and involvement of such antibodies in controlling the development of elevated autoimmunity. Auto reactive T cells and antibodies against various self tissues of the body has been shown to occur in aging B6 mice 34. Therefore, antianti-b6 antibodies produced in aging B6 mice may be induced by autoreactive T cells against self MHC molecules and be involved in suppressing the anti-b6 MHC that perhaps occurs in B6 mice. Antianti-self antibodies were also shown to be spontaneously present in the sera of autoimmune MRL mice strains, a model for systemic lupus erythematosus (SLE) disease 26. The presence and levels of anti-anti-self antibodies in MRL mice sera was shown to parallel the presence and levels of the autoantibodies typically associated with SLE26. Comparing the level of binding of biotinylated B6 anti-balb/c to the reciprocal Balb/c anti-b6 (Figure 3) to that of binding of biotinylated Balb/c anti-b6 to B6 anti-balb/c alloantiserum (Figure 2 a), we find that there is a higher level of binding in the latter. However, the result of antibody and complement mediated