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Isolation and characterization of the suppressor regulatory circuit controlling the immune response to… Steele, John Kevin 1986

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ISOLATION AND CHARACTERIZATION OF THE SUPPRESSOR REGULATORY CIRCUIT CONTROLLING THE IMMUNE RESPONSE TO THE MASTOCYTOMA P8L5 AND STUDIES OP CANCER IMMUNOTHERAPY BY SUPPRESSOR MODULATION  by  JOHN KEVIN STEELE B.Sc., University of Victoria, 1981  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MICROBIOLOGY  WE ACCEPT THIS THESIS AS CONFORMING TO THE REQUIRED STANDARD  The University of British Columbia July, 1986  « J. Kevin Steele, 1986  In p r e s e n t i n g  this  thesis  r e q u i r e m e n t s f o r an B r i t i s h Columbia,  it  freely  agree that for  understood that for  financial  Library  s h a l l make  for reference  and  study.  I  f o r extensive copying of  h i s or  be  her  g r a n t e d by  s h a l l not  be  The  (Vc/Vfr^vo  University  of  1956 Main Mall V a n c o u v e r , Canada V6T 1Y3  DE-6 C3/81}  British  of  further this  Columbia  thesis  head o f  this  my  It is thesis  a l l o w e d w i t h o u t my  permission.  Department o f  the  representatives.  copying or p u b l i c a t i o n  gain  University  the  s c h o l a r l y p u r p o s e s may by  the  the  I agree that  permission  department or  f u l f i l m e n t of  advanced degree a t  of  available  in partial  written  ii  ABSTRACT  This thesis is in three parts, each representing the progression of work over the 3 year period in which i t was done. centered on the B16G monoclonal antibody.  Initially, work  This antibody had been  previously raised by immunization of Balb/c mice with affinity-purified P815 tumor specific T-suppressor cell factor (TsF). Preliminary studies with B16G had indicated i t could pan out T-suppressor c e l l (TsC) populations from DBA/2 splenocytes, resulting in an enhanced mixed lymphocyte reaction (MLR), and could slow in vivo the growth of P815 or M-l tumors in DBA/2 mice.  Since the monoclonal appeared to bind to a  constant determinant of DBA TsC's and TsF's, a study (Chapter I) was undertaken to identify the factor to which B16G bound.  Bl6G-reactive  material was found to be isolatable from the membranes of DBA/2, MRL/lpr, and CBA mice splenocytes, as well as human tonsillar lymphocytes.  These  purified factors were found to be specifically suppressive in the MLR,  and  were major histocompatibility complex restricted, that i s , would not cross an H-2 (or HLA in humans) barrier.  Biochemically, these TsF's (or  solubilized TsC receptors) were found to have a native molecular weight of 80 kD (±10 kD) by gel f i l t r a t i o n , and to be readily dissociatable into 45 kD and 25 kD moieties as observed by SDS-PAGE analysis.  The  crossreactivity of B16G between murine and human proteins was fortuitous, but B16G is not apparently reactive with TsF from a l l mammalian species.  iii  For example, canine splenocyte extracts purified over B16G-4B columns resulted in no yield of TsF at a l l . Since B16G was demonstrated to be pan-reactive with DBA/2 TsC's, i t was used as a probe to isolate hybridomas producing T-suppressor factors specific for the P815 tumor system (Chapter II and III).  Two hybridomas  were isolated using this method; A10, which produces a TsF^ which i s i d , (binds to nominal P815 antigen) and specifically inhibits the +  response to P815 in vivo and in vitro (assayed by cytotoxic T-lymphocyte activity (CTL), and A29, which produces anti-idiotypic ( i d ) TsFg with -  similar inhibitory characteristics.  Both A10 and A29 TsF have a native  m.w. of 70,000, with 45 and 25 kD subunits, similar to the polyclonal TsF isolated before.  Both factors bind B16G and an antiserum raised to A10 ,  demonstrating their biochemical and serological similarity.  These 2  hybridomas, therefore, represent the hybrid analogues of the Ts^ and TSg suppressor population, and delineate the id/aid regulatory network controlling the immune response to the P815 tumor. The final part of the thesis (Chapters IV and V) demonstrates two methods of utilizing this information to evolve several approaches to cancer therapy by suppressor modulation.  Deletion of whole polyclonal  suppressor populations by administration of Bl6G-toxin conjugates in vivo was found to frequently cause complete tumour regression, even in mice with advanced tumor growth.  Mice so treated experienced long term  survival, with no apparent i l l effects or induction of autoimmunity  iv  induction.  Similarly, immunization of DBA animals with A10 TsF prior to  tumor dose was found to have a powerful protective effect against the growth of a variety of DBA/2 tumors, due to interference in the development of suppressor populations. Since B16G is demonstrably active against human suppressors, these approaches to cancer therapy might be applicable to the treatment of human neoplasms. The thesis, therefore, demonstrates the value of B16G as a tool for monitoring levels of suppressors, the profound impact that suppressors and their factors have on tumor growth, and the use of suppressor modulation therapies in treating cancer.  V  TABLE OF CONTENTS  PAGE Abstract  i i  List of Tables  viii  List of Figures List of Abbreviations Acknowledgements Introduction and Literature Review I. II.  IV.  xiii xv 1  Suppressor Background  1  Cancer Therapy by Immune Modulation  4  A. History of Tumor Immunology  4  B. Neoplastic Therapy Using Monoclonals to TAA's  7  C. Treatment of Neoplasias by Immune Modulation  9  1. Interleukin-2 and the LAK system  III.  ix  9  2. 11-1  11  3. Modulation of TsC by chemical administration  12  4. Anti-I-J therapy  12  The P815 Tumor System  13  Current Proposal  14  vi PAGE Chapter I - Isolation and Characterization of Polyclonal TsF from Murine Spleens and Human Tonsillar Cells Using Monoclonal Antibody Immunoadsorbence  16  Introduction  16  Materials and Methods  18  Results  24  Discussion  ...  Summary ...  44 47  Chapter II - Isolation and Characterization of a Tumor-Specific T-Suppressor Factor (TsF) from a T-cell Hybridoma  48  Introduction  48  Materials and Methods  50  Results  59  Discussion  78  Summary  -  83  Chapter III - Characterization of an Anti-idiotypic TsCg Hybrid Analogue Involved in the Regulation of the Immune Response to the P815 Mastocytoma  86  Introduction  86  Materials and Methods ..  88  Results  94  Discussion  109  Summary  116  vii PAGE  Chapter IV - Suppressor Deletion Therapy Using Bl6G/Toxin Conjugates  117  Introduction  117  Materials and Methods  119  Results  124  Discussion  132  Summary  136  Chapter V - The Therapeutic Potential of Purified Monoclonal TsF as an Anti-Tumor Vaccine  137  Introduction  137  Materials and Methods —  139  Results  142  Discussion  154  Summary  '•  157  Thesis Summary Literature Cited  158 '.  163  viii  LIST OF TABLES PAGE Table I  II  III IV V  VI  Effect of HT3F and B16G on a syngeneic MLR and on response to mitogen  33  Average tumor size of mice receiving A10 TsF and various tumors  74  Comparison of A10 to various antigen-specific TsFs  83  Summary of B16G-Hp experiments  134  Effect on tumor growth of active immunization with affinity purified A10 and control materials  151  Effect on tumour growth of active immunization with A10 or BW5147 cells  153  Ix  LIST OF FIGURES PAGE Figure 1 2  3  4 5  ELISA assay of various column eluates for polyclonal murine TsF  25  ELISA and protein concentration profiles of human tonsillar membrane extract eluted from B16G-4B immunosorbent  27  Results from MLR containing eluates from B16G or irrelevant monoclonal antibody immunoadsorbents (polyclonal MTsF)  28  MLR: biological activity of HTsF material purified over B16G  30  MLR comparing biological activities of HTsF isolated from two different donors  32  6  SDS-PAGE analysis of polyclonal mouse TsF  34  7  ELISA profile of material recovered from SDS-PAGE gels (mouse polyclonal TsF) SDS-PAGE analysis of human tonsilar extracts purified over B16G-4B  8A  36 37  8B  SDS-PAGE gel analysis of human polyclonal TsF  38  9  ELISA profile of B16G-4B column eluates fractionated over a G150 Sephadex column (mouse polyclonal TsF)  40  10  ELISA activity profile of purified HTsF fractionated over G150 Sephadex  41  11  Suppressive activity of MRL and CBA B16G eluates  43  12  Flow diagram illustrating the method used to isolate the A10 hybridoma  53  X  List of Figures (continued) PAGE Figure 13  Elution profiles of A10 and G10 culture supernatants from B16G immunoadsorbent columns  60  Elution profiles of A10 and BW5147 ascites passed over P815 Ag-4B adsorbent column  63  ELISA reactivity of A10 TsF purified over P815-4B columns  64  Growth of P815 tumors in DBA/2 mice Injected i.v. with 25.0 pg of A10  65  16B  Survival curves of mice treated with 20 yg A10 i.v. ..  66  17  In vivo activity of affinity purified A10 TsF in P815-bearing DBA/2 mice  67  18  Effect of different dose levels of A10 Bl6G-affinity purified material on the growth of P815 tumors in DBA/2 mice  69  14 15 16A  19  In vitro activity of A10 TsF in a chromium 51 release assay (CTL killing)  70  20  The effect of affinity purified A10 material on the MLR .  71  21  Antigen specificity of A10 TsF in vivo activity  73  22  Elution profile of A10 ascites over Sephadex G150  75  23  The effect of Bl6G-reactive fractions from G150-purified A10 TsF on the growth of P815 tumors  76  24  SDS-PAGE analysis of A10 TsF  77  25  ELISA reactivity of A10 T3F subunits eluted from SDS-PAGE Abrogation of A10 TsF suppressive activity by in vivo administration of B16G monoclonal  79  26  80  xi Lst of Figures (continued) List PAGE Lgure Figure 27  Flow chart describing the method used to analyse splenocytes of DBA/2 mice bearing P815 tumors for their relative levels of TsF, TsF^ and TsF2  95  28  TsC cell flux in tumor-bearing DBA/2 mice  96  29  A29 sandwich ELISA assay  98  30  Purification of A29 (aid TsF2) over A10 TsF-4B columns  99  31  SDS-PAGE analysis of affinity purified A29 TsF  32  Intracellular calcium flux within A29 hybrid cells stimulated with A10 TsF or B16G MAb Intracellular calcium flux within A10 cells stimulated with A29 TsF or B16G monoclonal  103  A29 TsF is suppressive in a Cr^l realease assay (CTL killing)  105  Panning of DBA/2 splenocytes over A29 TsF results in enhanced CTL responses  106  33 34 35 36  101 102  Growth of P815 tumor in mice receiving undiluted A29 TsF isolated from B16G-4B columns  107  37  Survival curves for Figure 36 (A29 TsF in vivo)  108  38  The P815 suppressor network  110  39  Structure of the hematoporphyrin molecule  123  40  The in vitro effect of B16G-Hp  125  41  The effect of RaMIg-Hp on A10 cells in vitro  126  42  Survival curves of P815 bearing DBA/2 mice  128  Li3t of Figures (continued)  xii  PAGE Figure 43 44 45 46  47  48  49 50  Growth of P815 tumor in mice receiving B16G-Hp treatment  129  Comparison of tumor growth in progressbr and regres3or mice receiving B16G-Hp therapy  131  Growth of P815 tumor in DBA/2 mice previously immunized with BW membrane extracts or A10 ascites purified over P815 Ag columns Growth of L1210 tumor in DBA/2 mice previously immunized with BW5147 membrane extracts or A10 ascites eluted from P815 Ag columns  143 144  Growth of P815 tumor in DBA/2 mice previously immunized with BW5147 membrane extracts or A10 ascites eluted from B16G-4B columns  146  Growth of L1210 tumor in DBA/2 mice previously immunized with BW5147 membrane extracts of A10 ascites eluted from B16G-4B columns  148  Survival curves of mice immunized with A10 or BW5147 ascites purified over B16G-4B columns  149  ELISA results from sera of Balb/c and DBA/2 mice immunized with affinity-purified A10  150  xili LIST OP ABBREVIATIONS ABA  -  p-azobenzenearsonate  A10  -  suppressor T c e l l hybridoma or i t s secretory product directed to the tumour P815  APC  -  antigen presenting cells  B16G  -  a monoclonal antibody against a suppressor factor derived from the heterogeneous suppressor T c e l l population  CFA  -  Complete Freund's Adjuvant  Con-A  -  concanavalin-A  CPM  -  counts per minute  CTL  -  cytotoxic T lymphocyte  DME  -  Dulbecco's modified Eagle's medium  DTH  -  delayed type hypersensitivity  ELISA  -  enzyme-linked immunosorbent assay  FCS  -  foetal calf serum, heated at 56°C, 30 min.  H-2  -  MHC of mice (MHC)  -  the idiotype of an immunoreactive molecule  anti-id (Id )  -  antibodies/factors directed to the idioptype (of A10 TsF)  Ig  -  immunoglobulin  11-2  -  interleukin-2  kD  -  kilodalton  KLH  -  Keyhole limpet haemocyanin  MHC  -  Major Histocompatibility Complex  Id (Id ) +  -  xiv Abbreviations (continued  pg  -  micro gram  jil  -  micro l i t r e  mm  -  millimetre  mM  -  millimolar  MLR  -  Mixed Leukocyte Reaction  M  -  Molar  MW  -  molecular weight  ng  -  nanogram  PBS  -  phosphate buffered saline  PMA  -  phorbol myristic acetate  RaMIg  -  rabbit anti-mouse immunoglobulin  rpm  -  revolutions per minute  SDS-PAGE  -  sodium dodecyl sulfate-polyacrylamide gel electrophoresis  ThC  -  helper T cells  3  [H]TdR  -  TsC  -  suppressor T cells  T3F  -  a factor secreted by TsC and capable of replacing TsC activity  3  H-labelled thymidine  ACKNOWLEDGEMENTS  The research atmosphere of Dr. Levy's lab is highly collaborative, and I am indebted to the entire lab for their assistance, and in some cases, their forebearance.  Specifically, I would like to acknowledge  Anthea Tench Stammers for her in vitro work with the suppressors, Agnes Chan for her gels and many control experiments, and Stephen Whitney, Daniel Liu, and Lily C-Wat for their work on heamtoporphyin therapy.  I  further thank Dr. Glen Taylor at Children's Hospital for making unlimited supplies of tonsils available for study, and Rosario Bauzon for patience and s k i l l in typing numerous papers and this thesis; and for Julia, my thanks for three years of superb direction and friendship.  1  I n t r o d u c t i o n O u t l i n e and L i t e r a t u r e Review TsC'a, T s F ' s , and Cancer  I.  Suppressor  Therapy  Background  In the decade o f the 1970's the e x i s t e n c e o f 2 d i s t i n c t a n t i g e n - s p e c i f i c r e g u l a t o r y lymphocytes positively  was e s t a b l i s h e d :  a population of  r e g u l a t o r y c e l l s r e f e r r e d t o as T h e l p e r c e l l s (ThC) ( 1 - 7 ) , and  a p o p u l a t i o n o f down r e g u l a t i n g c e l l s c a l l e d (8-20).  thymus d e r i v e d  T s u p p r e s s o r c e l l s (TsC)  A n t i g e n n o n - s p e c i f i c down r e g u l a t o r y c e l l s , ( f o r example the  p r o s t a g l a n d i n Eg producers f i r s t ( 1 2 ) ) , a r e independent  o f these  e x t e n s i v e l y d e s c r i b e d by G i r s c e l l i systems.  The presence o f suppressor c e l l s was f i r s t o b t a i n e d by Tada r e g a r d i n g the s p e c i f i c  suggested by the r e s u l t s  inhibition  f o r m a t i o n t o the p a r a s i t e a s c a r i s i n r a t s  o f IgE a n t i b o d y  (19) and by the work o f Gershon  et a l . , r e g a r d i n g t h e c e l l u l a r bases o f immune non-responsiveness ( 2 0 ) . These  i n i t i a l o b s e r v a t i o n s were q u i c k l y c o n f i r m e d i n o t h e r a n t i g e n systems  by o t h e r i n v e s t i g a t o r s  (21-24).  TsC's were i m p l i c a t e d as the cause o f  s p e c i f i c nonresponsiveness i n the response t o TGAL by B e n a c e r r a f and Kapp (11),  i n t h e r e g u l a t i o n o f IgE s y n t h e s i s by Yamamura (13), i n EAE ( 1 4 ) ,  neonatal tolerance In the l a t e P815  (15) and i n immune response r e g u l a t o r y networks ( 1 6 ) .  1970's K i l b u r n and Levy i d e n t i f i e d  mastocytoma system  (8,9,10).  tumor s p e c i f i c TsC's  L e c l e r c and coworkers  found  specific  s u p p r e s s i o n i n tumor b e a r i n g mice ( i n d u c e d by o n c o r n a v i r u s ) ( 1 8 ) . U r b a i n and c o l l e a g u e s demonstrated  i n the  Later,  that reduced tumor growth r e s u l t e d  a f t e r immunization o f mice w i t h TsC's ( 1 7 ) .  2  By the 1980's, the Network Theory of Jerne (25) had been expanded to include several competing theories regarding the interactions of id and anti-id receptor bearing suppressor cells leading to specific unresponsiveness.  The most enduring and generally applicable model was  advanced by Gershon and his colleagues (26).  In general, an inducer (Tsl)  TsC subset (bearing the c e l l surface markers I - J , Lyt l 2 ~ and +  Qa-1 ) is activated by nominal antigen. +  +  The inducer TsC functions by  activating a transducer TsC population (Ts2) via a soluble id-bearing factor (TslF). Lyt l 2 . +  +  The transducer TsC population bears I-J, is Qa-1  +  and  Its receptor is a-id (i.e. targeted to idiotype), as is  the Ts2F factor released by i t .  The target of activated transducers is  the effector TsC subset (Ts3) which bear I-J, Lytl~2 markers and +  id  +  receptors.  Effector cells can be stimulated by nominal antigen and  may act in concert with the inducer TsC's to produce the TseF which act upon and suppress the activity of helper c e l l populations. been frequently observed to be H-2  The TseF has  ("I-J" region) restricted (26).  Other  models of TsC network interactions are variations on this theme, as put forward by Cantor (27), Kapp (28) and Benacerraf (29). Soon after TsC's were identified, their soluble mediators were also identified.  These TsF's can replace TsC in their function, as originally  described by Taussig (30), Benacerraf (31) and Tada (32).  These TsF's  interact in regulatory networks, and serve to extend the effective radius of action of TsC's by participating in the Tsl-2-3 cascade. After these original observations, TsF's were identified in a number of other systems: in the immune response to synthetic amino acid copolymers such as GAT by Kapp (33), in glu-tyrosine synthetic polymers by  3  Benacerraf (34), in IgE synthesis regulation by Yamamura (35) in human T c e l l lines by Ballieux (36) and in the blocking of the anti-tumor response to P815 by Levy (37). The exact nature of these soluble regulators remains elusive; the TsF molecule remains unsequenced and the genes controlling i t s synthesis have yet to be identified; as well, the antigen receptor of the TsC seems to be separate from the molecular entities identified by Davis and Mak in 1984, as indicated by the results of Hood et a l . , which demonstrated the B chain of the T c e l l receptor was not rearranged in TsC hybridomas or, in some clones, was apparently deleted (38). To a large degree, a concensus among the various investigators as to what is known about TsFs has been reached.  A review by Kapp et a l .  extensively summarized what was known (39) in 1983, and i t is indicative of how l i t t l e the field has progressed, that no significant further information has been obtained since then. in this f i e l d are primarily two-fold:  The problems blocking progress  to date, no hybridoma or TsC line  produces more than a few yg/litre culture supernatant of isolatable TsF; in addition, the molecule i t s e l f is "programmed" to be labile, and is notoriously delicate to work with (39).  In fact, with the absence of  definitive biochemical analysis some investigators have questioned (though not in the literature) the existence of TsF, believing the observed effects to be artifactual or non-specific.  From those factors upon which  rudimentary biochemical analysis has proven possible there has emerged a common pattern; the molecule seems to have a native molecular weight in the region of 70,000 Daltons, and can be cleaved into 45 and 25 kiloDalton subunits or fragments.  The 45 kD subunit has been demonstrated to bear  4  id  +  determinants and serves as the antigen binding segment.  In a series  of elegant experiments, Taniguchi demonstrated that the property to induce the suppressive signal that inhibits ThC function is also carried on this chain (40).  The 25 kD moiety has been shown to carry I - J  and to be the source of H-2 restriction (41).  +  determinants  The 2 subunits can be added  separately in vivo, and can s t i l l function to suppress specific immune responses, an observation which led to the Gershon theory/concept that the one subunit serves as a "Schlepper" (carrier) moiety for the other, delivering i t to the target (26).  These 2 subunits may or may not  (depending on the reporting investigator) be joined by a disulphide bridge into the complete 70 kD TsF.  The primary amino acid sequence, tertiary  protein structure, and genes controlling the formation of TsF's are unknown.  II.  Cancer Therapy by Immune Modulation - Review  A.  History of tumor immunology - Tumor associated antigens and Immunogenicity The f i r s t attempts at cancer therapy by specific  occurred over a century ago (42).  immunization  In 1916, E. Tyzzer, a pioneer in  specific tumor immunology, was the f i r s t to compile a l l the contradictory findings in his review (43) of the subject. Tumor associated antigens (TAA's) were f i r s t studied by Loeb at the turn of the century; he discovered strain specificity in rejection or acceptance of transplanted tumor tissue (44).  Flexner and Jobleng  immunized rats with heat inactivated tumor tissue in 1907; they achieved  5  immunological enhancement and specific tumor regression (45).  These  experiments were improperly controlled and were followed by a half century of conflicting data. In 1952, Hauschka wrote a scathing (46) review of the work that had been carried out to that point.  He reported on 2 major defects in the  research: early experimenters worked with animals of unproven geneology, making i t impossible to differentiate between rejection mediated by normal alloantigens or tumor associated antigens (TAA's); and second, the tumors used in this studies had been maintained and passaged for long periods and were poorly characterized in terms of their antigenic character (loss of TAA's or passenger viruses) (46). In 1953, Foley worked with methylcholanthrene induced tumors in syngeneic mice, and successfully demonstrated specific antigenicity of transplanted tumors(47). pretreatment  Similarly, Prehn and Main in 1957 found that  of an inbred mouse with irradiated methylcholanthrene induced  tumor cells could protect against challenge with the same tumor cells, demonstrating the existence of tumor specific transplantation antigens (TSTA) and their ability to mediate rejection (48). In 1961, i t was discovered that polyoma virus induced tumors had common TSTA's (49), a situation opposed to chemically-induced tumors, a l l of which express unique TSTA's (55).  In 1965, carcinoembryonic  antigen  (CEA) was discovered by Gold and Freedman (50) demonstrating an additional class of tumor associated antigens (TAA).  Since then, additional examples  have been demonstrated (51,52,53). TAA's, such as CEA (a 200 kD glycoprotein) are detectable only by serology (antibody from a different  6  species) and not by any T-cell proliferative response since most are essentially non-immunogenic in the host (50).  In 1957, working with the  newly isolated P815 mast c e l l tumor, Dunn and Potter discovered that immunization of host animals with P815 cells was completely ineffective in slowing tumor growth (54). methylcholanthrene-induced  In the case of most other tumors (which often have a variety of TSTA's  within a single tumor) (56), TSTAs can be demonstrated via specific immunity imparted by immunization with tumor cells or resection of a growing tumor in the syngeneic system, thus establishing the immunogenicity of this class of antigens.  These have been demonstrated to  be modified H-2 antigens (57,58) in C3H lung tumors and some chemically induced sarcomas in mice.  In the case of P815 tumors, i t has been  demonstrated that TSTA's are distinct from MHC antigens (59). In general, TAA's are detected serologically with specific antibodies from the sera of animals immunized with irradiated tumor cells (60) , while TSTA's are detected by tumor rejection tests.  If one  acknowledges that tumor unique antigens in fact rarely exist, and that the "defined" TSTA's demonstrable in murine chemically-induced tumors are unique to this system then one is forced to question whether a majority of tumors are capable of generating an immune response in the host and i f so, what mechanisms are called into play for potential tumor rejection. Burnett's original surveillance hypothesis postulated rejection of tumors via their specific antigen as target for CTL or ADCC mediated rejection (61) . However, many violations of this theory have been noted, especially in terms of cancers developing in priveleged sites (62).  7  A possible way to view this situation is to regard the successful rejection of tumors as an exercise in autoimmunity (see thesis Discussion, p. 159).  Since TAA's are usually, the result of abnormal expression of  normal genes, any successful anti-tumor response would of necessity override the normal regulatory mechanisms "designed" to prevent any self-responses in the animal.  The mechanisms of self tolerance are also  unknown, but there are currently three major hypothesized mechanisms; clonal deletion, clonal anergy, or specific suppression (63). a l l three mechanisms operate concurrently.  Possibly  An animal may be unable to  mount an immune response to a given tumor antigen as a result of "holes" in the specific CTL and the T helper cell repertoire programmed during thymic and post-thymic maturation, or any anti tumor response could be overriden by development of specific TsC populations.  This view of  tumorgenesis could account for the intense stimulation of TsC populations observed in the P815 system and others (10,18).  B.  Neoplastic therapy using monoclonal antibodies specific for cancer TAA's Acknowledging the possibility that some tumors have unique  antigens that can be detected serologically, how can this property be used as an approach for cancer therapy?  Anti-tumor antibodies have been  demonstrated to be of some value in the treatment of some leukaemias, and certain lymphomas (64).  These examples u t i l i z e lymphocyte markers (T  c e l l , B c e l l idiotypes) as TAA and thus represent systems which cannot be applied easily to other neoplasms.  In human clinical t r i a l s , anti-tumor  antibodies have met with l i t t l e success , and frequently give rise to  8  negative results, especially in the case of solid tumors in which instance i t is suggested that the antibody blocks access of CTL's to the tumor antigens (65). For such an approach to work, many criteria must be met: the tumor cells must a l l express the antigen in question; the antigen in question must exist, that i s , there must be a defineable TAA or TSTA, something that many tumors demonstrably lack; the antibodies used must be specific for the tumor antigen with minimal cross-reactivity to normal cellular antigens; the tumor specific antigen must be readily accessible to antibody; the tumor load i f too high cannot be dealt with and solid tumors are not easily accessible to CTL or antibody; and last, the tumor must lack the ability to modulate antigen; that i s , modify, or stop making the Ag once under immunological "attack".  If a l l these criteria are met,  the approach might be feasible, but in fact is rarely of any efficacy even in experimental systems (66). In mouse experimental systems, positive results with this approach have only been achieved with ascitic tumors (not solid) and only then i f antibody was given simultaneously with the cancer c e l l dose, a situation hardly analogous to human therapy (67).  Blood borne tumors are  more susceptible to this approach than other types (e.g. leukaemia) (68). Even in the case of leukaemia, c e l l numbers will i n i t i a l l y drop in response to the attack of the specific antibody, but soon rise in number due to antigen modulation in the target cells; (e.g. the cell surface antigens of certain lymphocytic leukaemias) (69) and the antigen i s reexpressed after the antibody is cleared. On the positive side,  9  successes in treatment of B cell lymphomas with anti-idiotypic antibody have been observed (70).  In an attempt to heighten the "punch" of the  specific Ab's, and in the tradition of the "magic bullet" approach proposed by Paul Ehrlich (71), various materials have been conjugated to the anti-cancer antibody.  These include r i c i n (72), diptheria toxin (73),  enzymes such as phospholipase C (74), or porphyrins (75-84).  C. Treatment of neoplasias by Immune modulation  1.  Interleukin-2 and the LAK system. Lymphocytes undergo "triggering"; that i s , the initiation of  DNA synthesis and division when their receptors interact with antigen. The carrying forward of this process to meaningful function is mediated by lymphokines or growth factors, of which Interleukin-2 (11-2) i s the most studied (85).  11-2 functions by mediating the G-j. -» S,G switch 2  (induction of proliferative phase) in T cells; i t i s a single protein of MW 15,420 Daltons.  Investigators  have, in the past 4 years, defined i t s  cDNA nucleotide sequence (86), and i t s amino acid sequence (87). Monoclonal antibodies are also available, and can abrogate 11-2 function (88). Recently, i t was observed that 11-2 i s responsible for maintaining some leukaemic neoplasms.  It has now been conclusively  demonstrated that some retrovirus induced tumors in gibbons are dependent on 11-2 for growth (89).  On the positive side, 11-2 mediated modulation  of the immune system appears to be potentially a powerful tool in cancer therapy.  10  A number of reports have demonstrated the value of in vivo administration of 11-2 in inducing anti-tumor responses, and in enhancing the anti-tumor effectiveness of adoptively transferred, long term cultured T-cells directed to the TAA's of the tumors involved (90,91,92).  Such  methods are of limited practicality in human cancer therapy, due to the difficulties of defining TAA's in relatively weakly immunogenic human tumors, and to the technical problems in maintaining and raising anti-tumor T-cell lines in humans (93). As yet, in addition, there is s t i l l no conclusive proof that T-cell mediated reactions to TAA's in human cancers leads to tumor destruction (94). An alternate method has been pioneered by Steven Rosenberg and his colleagues at Bethesda.  The method is called "adoptive  immunotherapy", and involves the induction of LAK (lymphokine activated killer) cells with recombinant Interleukin-2 (RI1-2) (95). The LAK system is not raised to TAA's, and is independent of both the NK ("natural k i l l e r " ) or CTL systems (96,97).  LAK cells are generated in vitro by  incubating fresh PBL's (peripheral blood leukocytes) of tumor bearing patients with RI1-2.  LAK can also be generated in vivo, at least in mice,  by systemic administration of RI1-2 (98). LAK cells thus generated when returned to the animal can destroy a very broad range of transformed and malignant cells, while having no effect on nearby normal tissues (96). Obviously, LAK cells are more easily made than specifically sensitized anti-tumor cells, especially since the advent of recombinant  DNA  technology has provided "unlimited" quantities of RI1-2 for use (99).  11  In a mouse model system, Rosenberg et al^. used adoptive immunotherapy on C57B1/6 mice injected intravenously with syngeneic MCA-105 tumor cells.  Animals were treated with adoptively transferred  C57/B16 or DBA/2 LAK cells and multiple RI1-2 injections (100). As followed by the obvious elimination of pulmonary MCA-105 metastases, this treatment was very effective. DBA/2 LAK cells were found to be as effective in conferring cancer resistance as B6 cells, demonstrating the absence of allogeneic restriction in LAK function, a finding which further demonstrates the possible value of this procedure in human therapies (95). Recently, 11-2 administration in vivo in Phase I clinical trials failed to enhance immunity in a l l patients tested save one, who demonstrated increased CTL and NK activity. U/M2 11-2 were administered,  To achieve this result 10,000  and a l l patients involved developed anti 11-2  IgG titres which were neutralizing in their effect (101).  2. 11-1. Anti-tumor studies in humans with Interleukin~l are very rare to date, and despite the extensive information available on i t s DNA and amino acid sequence, limited information on its mode of anti-tumor action is available. However, i n one study with human cancer, 11-1 administration was found to promote human monocyte-mediated toxicity directed toward the human melanoma line A375.  11-1 used for this  experiment was laboriously purified by HPLC (102).  12  3•  Modulation of TsC by administration of chemicals. Many chemicals and potions have been plucked from the shelf  and pressed into tumor "therapy".  While most of the chemicals commented  on here were selected as chemotherapeutic agents because of their apparent and somewhat selective cytotoxicity for tumor cells, they have in retrospect been shown, at some levels, to also have a selective effect by preferentially k i l l i n g TsC. cyclophosphamide  (103).  The most widely used of these has been  Nitrosoureas have been used as well for many  years in the treatment of human neoplasms (104).  Recently, these  chemicals (e.g. BCNU) have been demonstrated as being specifically toxic to TsC's, and this has been conclusively anti-tumor protection (105).  proven as their mode of  These drugs have a very narrow window  between benefit and systemic toxicity (due to their efficiency at alkylating and carbamylating normal cells) (106), but for some cancers, such as childhood leukemia, remain the most effective form of therapy available.  4.  Anti-I-J therapy. T-suppressor cells appear to bear I-J "MHC"  antigen markers,  at least in those strains in which a l - J antisera can be raised. a l - J antiserum can specifically k i l l suppressor cells in the appropriate strains.  Administration of al-J-allo antiserum to tumor-bearing animals  results in a reduction of tumor growth for this reason (107).  Similarly,  when a l - J monoclonals became available, they also proved effective in interfering with tumor growth in the appropriate murine strains (108).  13  While clearly functional, there are two clear problems associated with this approach.  First, I-J antigens have yet to be defined, and with the  nucleotide sequencing of the entire murine MHC region are doubted by some to exist at a l l .  Second, a l - J reagents cannot be raised in nearly a l l  strains of inbred mice, let alone humans.  III.  The P815 Tumour System The i n i t i a l observation that injection of P815 tumor cells in  DBA/2 mice induced the formation of P815-specific TsC's by day 8 was made by Takei et a l . (109) in 1978.  These TsC's were found to specifically  inhibit the in vitro generation of P815 specific CTL's (8,9).  From these  <xP815 TsC, a soluble tumor specific TsF could be isolated by sonication and immunoadsorption; these could replace the TsC's in the in vitro CTL assay i f added no later than 30 h into the assay (110).  The molecular  weight of this TsF was tentatively identified at 40-60 kD, with a PI of 4.6-4-9. The Ag-binding capability of the factor could be demonstrated by Its capacity to be adsorbed over P815-immunoadsorbent columns, and not over L1210 or RAMIg-columns, demonstrating i t s antigen specificity and lack of identity with Ig (8,9,110).  It was observed that both TsC and TsF  could be obtained from mice primed intraperitoneally with either killed P815 cells or P815 membrane fragments (109).  The TsC and TsF obtained  thus bore Ia determinants, and were found to be non MHC restricted (determined using radiation chimeras of a different MHC)  (110).  The TsC  capable of inhibiting the response to P815 was determined to exhibit the  14  Lyt l 2 ~ phenotype; i t s apparent target was a Lyt l ~ 2 3 P815 +  +  +  specific CTL population. Antisera raised against TsF purified over P815-immunoadsorbent columns was found to react with both TsF and TsC but not CTLs; administration of this serum in vivo concurrent with P815 tumor injection resulted in a significant slowing of tumor growth (111). Finally, a monoclonal antibody, B16G, was raised against affinity-purified aP815 TsF and was found to have similar characteristics of reactivity and function as the antiserum described above (112).  IV.  Current Proposal  In this study, B16G was used as a probe to further study TsF's and their relevance to the regulation of the immune response to a tumor. In Chapter I, i t is demonstrated that functional polyclonal TsF's can be isolated, using B16G as an immunoadsorbent, from the spleens of DBA/2 and CBA mice (120).  Similarly, functional polyclonal TsF can be isolated from  human tonsilar lymphocytes  (121).  These results indicate the pan-reactive  nature of the B16G monoclonal, and reveal, by gel f i l t r a t i o n and SDS-PAGE, the native MW of the TsF molecule to be in the region of 70 - 80 kD with 45 kD and 25 kD subunits, as predicted by other investigators (see above).  This technique of polyclonal TsF isolation was used to  demonstrate that the spleens of autoimmune MRL/lpr mice contain normal levels of TsF, thus implying their autoimmune defect to be of a monoclonal, rather than polyclonal suppressor nature.  15  In Chapter II, the Isolation and in vivo function of a monoclonal aP815 TsF-producing T-cell hybridoma (raised using B16G as a probe) is described (136). In Chapter III, the isolation of a second order aid TsF^ producing hybrid in the P815 system is described. A theoretical model of TsC and contrasuppressor subpopulation interactions in P815 Immune regulation is put forward. In Chapter IV, a novel form of cancer therapy is demonstrated, using Bl6G-hematoporphyrin conjugates to specifically delete TsC populations from tumor bearing mice.  This approach uses immunotoxlns  targeted to regulatory T cells and is not subject to the limitations inherent in "magic bullet" approaches where the antibody is directed to cancer-"specifie" antigens. In Chapter V, an autoimmune reaction to self TsF stimulated by immunizing aimals with anti-P815 TsF produced by the hybrid line described in Chapter III was shown to induce immunity to suppressor factor.  This  procedure was found to be a powerful therapeutic measure both in P815 tumors and in the unrelated tumor L1210 (122). The final chapter discusses the potential of using this approach of immune regulator cell modulation as a method of cancer therapy.  16  Chapter I  Isolation and Characterization of Polyclonal TsF from Murine Spleens and Human Tonsillar Cells Using Monoclonal Antibody Immunoad3orbence  INTRODUCTION  T-suppresspr cells and their soluble counterpart (TsF's) directed to, and controlling the response towards the mastocytoma P815 in DBA/2 mice had been identified and characterized by work done in our laboratory in the late 1970'3 (8,109-111).  The P815 antigen-specific TsC population was  found to be Lyt l 23 ; and to act by preventing the generation of +  anti-tumor CTL's in an in vitro P815 k i l l i n g assay (8). Levels of TsC were found to peak 10-12 days after subcutaneous injection of P815 cells in vivo, as determined by the above assay; at this time, tumor growth proceeded rapidly, with fatal metastases (110).  From these TsC, a soluble  factor (TsF^) could be isolated by sonication, and subsequent purification over P815 immunoadsorbent columns.  This factor could  functionally replace i t s parent TsC'3 using the in vitro assay (111). Antisera raised to this factor in either syngeneic or allogeneic animals were found to inhibit the growth of P815 tumor cells in vivo (2), and to block the activity of tumor-specific TsC in vitro (111). Subsequently, from affinity purified TsF immunized Balb/c splenocytes fused to NS-1 cells, a monoclonal hybridoma, B16G, was raised. The hybridoma secretes a monoclonal antibody (MAb) which has properties  17  analogous to the aTsF antiserum; in vivo, administration of B16G was found to slow the growth of P815 tumor cells in mice, and to significantly enhance survival (111). The B16G monoclonal was tested for specificity to the P815 tumor system "by assessing its effect on an unrelated tumor syngeneic to DBA/2 mice, Ml.  Interestingly, B16G was found to significantly slow the growth  of Ml cells in vivo as well.  This observation opened the possibility that  the Bl6G-monoclonal was recognizing a determinant of the TsF/TsC receptor common to DBA/2 TsC regulatory cells.  Bl6G-reactive cell surface  determinants were clearly demonstrated to be present on those cells which function to down-regulate the MLR by experiments in which panning (removal of Bl6G-reactive DBA/2 cells) of DBA/2 splenocytes over Bl6G-coated plates resulted in an enhanced MLR (112). Thus, i t appeared that B16G was recognizing an epitope of the TsC receptor and i t s released soluble TsF molecule common to a l l DBA/2 Ts cells.  Whether or not this epitope was unique to DBA/2 cells or shared in  common with other strains or species was not clear, since B16G panning MLR experiments performed with Balb/c splenocytes were inconclusive. In the following series of experiments the molecule with which B16G reacts was isolated by immunoadsorption of lymphoid extracts over Bl6G-immunoadsorbent columns.  Functional TsF (i.e. able to suppress  allogeneic MLRs) could be obtained from both murine and human lymphoid extracts.  The biochemical characteristics of this molecule, and i t s  relationship to T-cell receptors and TsF's described by other investigators, is discussed.  18  MATERIALS AND METHODS  Experimental Animals.  DBA/2, CBA, MRL/lpr, B10-BR Balb/c and (Balb/c x  CBA)F^ mice were used between the ages of 8 and 12 weeks of age. were purchased from the University of British Columbia breeding  Monoclonal Antibodies.  They facility.  The production and nature of the B16G monoclonal  antibody (subclass y2a) has been described previously (112);  Large  quantities were prepared by growing the hybridoma as an ascites tumor in Balb/c or (Balb/c x CBA)F^ mice.  The MAb was precipitated from ascitic  fluid with 50% saturated (NH ) S0^, dissolved in PBS and 2  against the same buffer.  An irrelevant MAb  dialy3ed  (directed to human lung  proteins) was treated in the same manner.  Immunoadsorbents. irrelevant MAb  Immunoadsorbents were prepared by linking B16G or an  to cyanogen bromide activated Sepharose 4B beads according  to standard methods (111).  Immunoadsorbents were stored at 4°C in PBS  plus .02% sodium azide.  Preparation and Purification of T Suppressor Factor (TsF). mice were sacrificed by cervical dislocation.  Sixteen DBA/2  Their spleens were removed  and passed through a #16 gauge mesh into ice cold PBS.  Spleen cells were  sonicated 5 x 30 second bursts in an ice bath, and their membrane proteins solubilized with lysis buffer (.14 M NaCl, 1.5 mM MgCl  2>  10 mM Tris-HCl,  and .1% NP40). A soluble membrane extract was prepared by centrifugation  19  of the lysate at 10,T00xg at H C for 30 min.  The supernatant contained  TsF as described previously (111). A l l subsequent procedures were carried out at 4°C.  The cell lysates were absorbed by passage over 10 ml  immunoadsorbent columns containing either B16G or an irrelevant MAb.  The  method involved "cycling" of the sonicate over the absorbent column three times followed by incubation of material on the column for 30 min.  The  columns were then exhaustively washed with PBS until the 280 nm absorbence of the wash approached 0.00.  Elution was performed with ice-cold 0.1N  HC1; 1.0 ml fractions were collected and immediately neutralized with 1.0 M Tris buffer, pH 7.0.  The fractions eluted were monitored for 280 nm  absorbence or by ELISA (see below) for the presence of TsF. Equivalent DBA/2 splenocytes cycled over an irrelevant MAb immunoadsorbent column served as controls.  ELISA.  The enzyme linked immunosorbent assay (ELISA) was used at various  stages in these studies to monitor for TsF.  Materials to be analyzed for  the presence of TsF (i.e. B16G-reactive material) were diluted in carbonate coating buffer (pH 9.6) (dilutions were titrated for individual tests) in 100 y l aliquots applied to Dynatech Immulon I plates and incubated overnight at 4°C.  Plates were washed three times with  PBS-Tween (PBS saline + .05% Tween-20) between each of the following steps.  The plates were f i r s t blocked for 30 min at 25°C with 100 yl  unconjugated rabbit anti-mouse Ig (20 yg per ml diluted in PBS-Tween). B16G MAb was then reacted at 20 yg/ml in 100 y l aliquots to each well for 1 h at 25°C.  Plates were then developed with alkaline phosphatase  20  labeled rabbit anti-mouse Ig and Sigma substrate.  Plates were read at 30  and 60 min on a Flow Titertek at 405 nm; appropriate controls were run simultaneously on the same plate.  Murine Mixed Leukocyte Reactions (MLR). The biological activity of various TsF preparations were monitored by use of the MLR, since i t was shown previously that MAb B16G appeared to react not only with the P815-specific TsF but also with other regulatory molecules, and that i t influenced the outcome of the MLR when cells were treated in vitro with B16G (112).  Splenocytes from DBA/2 mice were cultured with various  concentrations of TsF or appropriate control material in RPMI 1640 containing 10% FCS in Falcon microtiter wells (#3072) at a concentration 5 of 3 x 10 cells/well. Irradiated splenocytes from B10.BR mice (1000 5 rads) were co-cultured as stimulators at a concentration of 3 x 10 cells/well in a total volume of 200 y l . Cultures were incubated at 37°C for 72 h in a humidifed 37°C incubator at 5% C0 . 2  At that  time, 50 y l of H-thymidine (20 pCi/ml) in RPMI 1640 was added to 3  each well.  Cells were harvested 18 h later on a Mash harvester and 3  counted for 30 sec using the H window on a Packard Tri-Carb 4550 scintillation counter (United Technologies). Preparation of HTsFs. Tonsillar material was obtained from Dr. Glen Taylor at the Children's Hospital (Shaughnessy Site, Vancouver, B.C., Canada). Tonsils were sliced in sterile PBS saline, passed through a fine nylon mesh and  21  finally reduced to single cell suspensions by repeated passage through a 26 gauge needle.  Cells to be used in MLR (4 x 10  and washed repeatedly in warm (37°C) PBS.  7  total) were removed  The remaining cells, to be Q  used for extraction of HTsFs (about 1 x 10 ) were solubilized by suspension in NP40-containing lysis buffer at 0°C (.5% NP40, 10 mM Tris, 1.5 mM MgCl and 0.14 M NaCl, pH 8.6), and the HTsF they contained were 2  purified by immunoadsorption over Bl6G-columns. Human Mixed Leucocyte Reaction (MLR). HTsF purified from tonsil extracts over B16G Sepharose immunoadsorbent columns was evaluated for biological activity using an allogeneic mixed leukocyte reaction (MLR). Whole tonsils (removed from donors about 1 hour previously) were washed repeatedly in sterile PBS, separated to a single cell suspension, and further washed by 7  centrifugation in sterile PBS. 4 x 10  lymphocytes were resuspended in  DME/20% FCS for use in the MLR, while the remaining cells (about 10 ) 9  were lysed for extraction of TsF over B16G-4B columns (see above). Cells used as responders in the MLR were centrifuged, and resuspended in DME at a concentration of 2 x 10  cells/ml.  1 x 10  cells in 500  yl DME were applied to each of 40 2.0 ml wells (Linbro Tissue Culture Plates #76-033-05).  Cell serving as allogeneic targets (i.e. tonsils from  a different donor) were irradiated with 800 rads, and applied at a 1:1 stimulator to responder ratio (in 500 yl DME) to the 40 2.0 ml wells. Control wells were also set up containing responder or stimulator cells only.  Each 1.0 ml fraction eluted from Bl6G-columns was dialysed for 18 h  o against DME at 4 C, sterilized by Millipore f i l t r a t i o n and then 100 yl  22  of each applied to the test wells in either triplicate or quadruplicate. Fractions eluted from control irrelevant MAb  columns (anti-human lung Ag)  were processed in the same manner and applied to control wells.  As an  additional control, HTsF materials purified from one set of tonsils were tested for activity against both allogeneic and syngeneic responder cells.  For studies in which B16G MAb was added in order to neutralize  TsF, 100 jig B16G, 0.  dialysed against PBS, was added to each well on day  To test the effect of TsF on mitogen-stimulated responders, 10 yg in  10 y l medium phytohemagglutinin (PHA) was added to 10^ responder cells on day 0, in the presence or absence of 30 yg HTsF. incubated for 72 h at 37°C and in 10% C0 , 2  Plates were  then 50 y l of  3 H-thymidine (NEN) containing 100 yCi was added to a l l wells.  Cells 3  were harvested 24 hrs later on a mash harvester and counted for  H  incorporation on a Packard Tri-Carb 4550 scintillation counter (United Technologies).  Titrations of individual fractions eluting from  Bl6G-Sepharose columns were run at least in triplicate. Sephadex Fractionation.  Materials eluting from immunoadsorbent columns  were further fractionated over a standardized Sephadex G-150 20.1 cm).  column (2.5 x  Materials to be fractionated were added in volumes not  exceeding 2.0 ml in 15% glycerol, and were run at a constant rate of 15.0 ml per h at 4°C.  Usually T3F preparations were cofractionated with  proteins of known molecular weights as standards (including bovine serum albumin (BSA) and myoglobin). fractions were collected.  Columns were run in PBS and 2.0 ml  Individual fractions or pooled samples (when  23  indicated) were dialysed against dH^O, and lyophilized prior to subsequent analysis by polyacrylamide gel electrophoresis (PAGE) or ELISA.  The column was calibrated prior to use with Blue Dextran (150 kD+)  BSA (68 kD), ovalbumin (43 kD), myoglobin (17.5 kD) and tryptophan (<1 kD).  PAGE. In most instances in this study, SDS-PAGE was carried out using standard procedures.  Briefly, 10% gels were used and developed by using  Wray's silver stain.  To prevent the development of staining artifacts  induced by 2-mercaptoethanol, reductions were carried out with 1% dithiothreitol, 2.3% SDS, 15% glycerol and 12.5% Tris HC1, pH 6.8. In this study, dithiothreitol was found to cause fewer artifacts than 2-mercaptoethanol.  Also, a l l solutions (running buffer, acrylamide) were  millipore filtered and a l l developing solutions were degassed before use. In order to enhance visualization of low protein concentration bands, thin gels (0.75 mm) were used. Materials to be run on PAGE (eluates from affinity columns or fractions from Sephadex columns) were lyophilized overnight at -50°C, following which they were dissolved in 100 y l of reduction mixture (60 y l of distilled HgO, 40 y l reduction mixture), applied to a 5% stacking gel and run at 50 V (20 amps) for 6 h.  In one instance,  materials were eluted from a stained PAGE. The gel was sliced into 3trips covering the range of 10-15 KD molecular weight. soaked for 48 h at 4°C in 500 y l d i s t i l l e d water.  Individual strips were 100 yl aliquots  of each 500 yl sample were diluted 1:2 in ELISA coating buffer and applied to ELISA plates for assay with B16G.  24  RESULTS  I.  Biological Activity of Bl6G-binding Material  It was demonstrated previously that immunoadsorbent columns prepared with B16G were capable of removing TsF from DBA/2 membrane splenocyte extracts as evidenced when f a l l through materials from a B16G column were discovered to have reduced for suppressive activity in the MLR (112). In order to determine whether B16G columns could be used to purify TsF, membrane extracts from splenocytes of groups of 16 DBA/2 mice were passed over B16G columns.  Equivalent material was passed over a column  containing an irrelevant MAb of the same isotype.  Elution was carried out  with 0.1N HC1, 1.0 ml fractions were collected and immediately neutralized with 1.0 N NaOH. Aliquots of each fraction (100 yl) were diluted 1:2 in the ELISA coating buffer, plates were coated and tested in quadruplicate for the presence of material reactive with B16G. Plates were read at 30 and 60 min.  Typical results are shown in Figure 1 in which i t can be seen  that B16G reactive material eluted in the acid fractions from B16G columns.  The specificity of this reaction Is shown by the absence of  significant levels of reactive material eluting from the irrelevant column.  It should be pointed out, that even when large amounts of extract  (16 spleen equivalents) were used, very l i t t l e measurable protein (by 280 nm absorbence) material was eluted from the B16G columns. Absorbance at 280nm from peak tubes rarely exceeded 0.100 and thus the specific material constituted considerably less than 0.1% of the starting material.  Total  FRACTION  NUMBER  Figure 1. ELISA of various column eluates for polyclonal murine TsF. ( •  • ), elution profile of material recovered from sensitized  spleen sonicates passed over a B16G-4B column; ( 0 unsensitized  3pleen  0 ), eluates of  extract passed over B16G-4B column;  eluates of sensitized  3pleen  (  flr  A ),  extracts passed over an irrelevant MAb-4B  column. Mice were sensitized by injection of 100 yg P815 membrane extract i.p. 4 days prior to sacrifice, a procedure demonstrated to boost levels of TsC.  Fractions of 1.0 ml volume were collected and assayed.  26  yield of TsF was measureably enhanced by sensitizing the animal with 100 jig P815 membrane extract i.v. 4 days prior to sacrifice; such a protocol is known to stimulate the development of TsC's. Figure 2 illustrates results obtained when extracts from human tonsillar material were passed over and eluted from either a B16G or an irrelevant immunoadsorbent column.  It can be seen that when assessed for  either 280nm absorbing material or ELISA reactivity only the Bl6G-Sepharose column retained reactive material.  These results indicate  that TsF reactive with B16G is present in human lymphoid tissue, although the total yield, as indicated by 280nm absorbing material, is not high. Further experiments were carried out to determine whether material eluting from B16G columns had retained biological (immunosuppressive) activity.  Pooled eluted material from a B16G column, and equivalent  material from an irrelevant column were titrated in the MLR for suppressive activity.  In the case of mouse TsF, a total of sixteen  spleen-equivalents was used in each experiment.  In duplicate sets of  MLRs, B16G was added to a final concentration of 50 pg/ml in order to determine whether any observed suppressive effect could be neutralized by the B16G MAb.  The results (Figure 3) show clearly that suppressive  material was eluted from the B16G column; the MLR was inhibited 100% at a 1:4 dilution and much of this inhibitory activity was abrogated when B16G was added to these MLR cultures.  Comparable dilutions of eluates from an  irrelevant column however, inhibited the MLR by only 37% and to these the addition of B16G had no effect.  These results thus demonstrate that a  Figure 2• ELISA and protein concentration profiles of human tonsillar membrane extract eluted from B16G-4B immunosorbents.  0D _ of tonsil oo  Q  extracts derived from 10 tonsillar cells passed over B16G ( • — • - • ) ; 0D  280 °^  t o n s : i  -  l l a r  extracts passed over irrelevant monoclonal column  (same subclass IgGl) ( 0from B16G-4B; ( 0 immunoadsorbents.  0 ). ( •  O ), 0D^  Q5  § ), 0D^  05  of fractions eluted  of fractions eluted from irrelevant  The x axis represents fractions eluted from the  immunosorbent columns. with substrate at 22°C.  ELISA is shown after 90 minutes of development  Figure 3. Results from MLR containing eluates from B16G or irrelevant monoclonal antibody immunoadsorbents.  Spleen extracts from 16 DBA/2 mice  were passed over the respective immunoadsorbent columns. Aliquots of the eluates were titrated in an MLR in which untreated DBA/2 and irradiated (1000 rads) BIO.BR spleen cells served as responders and targets, respectively.  In some wells, A 50% (NH^gSO^ cut of B16G was added  to B16G eluates ( • ( •  • ) at 48 jjg/ml. MLR of B16G eluates  "41 ) , or irrelevant eluate ( 0  0 ). MLR of irrelevant  eluates plus a 50% (NH^SO^ cut of B16G ( •---§ ). First data points represent undiluted TsF. than 10% in each case.  The standard error of the mean was less  In each individual experiment, groups were  performed in octupllcate. The whole experiment was repeated 5 times.  29  polyclonal TsF Is adsorbed by B16G, elution with 0.1N  that i t s activity is retained after  HC1, and that its activity in vitro is abrogated by  B16G.  In order to determine whether Human Bl6G-reactive material had immunosuppressive properties analogous to murine TsFs isolated in this way,  its effect on the MLR was tested.  Cells from the same tonsil from  which individual HT3Fs were prepared were cultured for a 24 h period, during which the syngeneic HTsF was affinity purified and dialysed, prior to exposure in culture to irradiated allogeneic cells in the presence or absence of the HTsF.  Initially, individual fractions eluted from B16G  columns were tested for their effect on the MLR.  A representative result  is shown in Figure 4 in which i t can be seen that eluted materials are suppressive and that suppression of the MLR correlates absolutely with the ELISA B16G reactivity of individual fractions. Thus, those fractions containing the highest concentrations of B16G-reactive antigens produced between 60 and 75% inhibition of the MLR.  Equivalent fractions taken from  an irrelevant column did not cause significant suppression. therefore indicate that the MAb B16G  These results  recognizes a suppressor determinant  which constitutes a constant region on both murine and human TsFs. In order to determine whether these HTsF were active across the major histocompatibility complex (MHC), the following experiment was carried out.  Tonsillar materials from two individuals were obtained on the same  day, a portion of the lymphoid cells from each donor was cultured, and from the remaining material HTsF was extracted, affinity purified, dialyzed, and tested the following day for suppressive activity in the one-way MLR.  The results are shown in Figure 5.  The HTsF, which is  80  0.5  M L R : biological activity of HTsF material purified over  Figure 4. ( o  -O  ),  OD^QJ  B16G.  of fractions eluted from B16G-4B; vertical columns  ( • ), suppressive activity of pooled fractions in the M L R .  Pooled  fractions to be tested in the M L R were dialyzed against D M E at 4°C, and 100 pi were added to an allogeneic M L R in which the responder cells were the same as those from which the HTsF was derived.  ( 0 ), suppressive  activity of a l l fractions eluted from an irrelevant immunoadsorbent (murine Ab of the IgGl subclass).  31  active on the syngeneic cells from which they were derived, have l i t t l e or no effect on allogeneic responder cells, indicating the effects observed with MLR are not nonspecific or due to lymphotoxins.  The results also  show that HTsF does not act as well on allogeneic cells, and thu3, is MHC (HLA) restricted.  Further experiments to determine whether the HTsF was  suppressive in nonimmunologic assays were carried out; i t s effect on the PHA mitogen response was assessed.  The results are shown in Table I, in  which i t can be seen that this material (HTsF), although inhibiting the MLR,  has essentially no effect on the PHA response of syngeneic cells. To demonstrate that the material responsible for the observed  suppression in the MLR was B16G cultures was investigated.  reactive, the effect of adding B16G  to MLR  Table I also demonstrates that the presence of  B 1 6 G in cultures abrogates the suppressive effect of HTsF, especially at lower HTsF concentrations  (3 to 10 yg), whereas the addition of  irrelevant murine IgG monoclonal had no significant effect.  II.  Biochemical Characterization of B16G-Reactive Material  Experiments were carried out to characterize the TsF-enriched  eluates  on PAGE. Eluted materials were lyophilized and run on gels adjacent to dilutions of the fall-through materials from which they were derived.  The  results for murine TsF (Figure 6 ) demonstrate a number of features. Materials eluted from B 1 6 G columns yielded clearly discernable bands in the region of 45 kD molecular weight, as well as a fainter band at about 25 kD.  32  XX!  I  Figure 5.  COLUMN  FRACTION  -*  5  MLR comparing b i o l o g i c a l a c t i v i t i e s o f HTsF i s o l a t e d from two  d i f f e r e n t donors.  ( 0-  1  0 ), s u p r e s s i v e  a c t i v i t y o f "A" HTsF a c t i n g on  "A" responders i n an A v s . B a l l o g e n e i c MLR; ( • a c t i v i t y o f "B" TsF a c t i v i t y ( • - - - • ) ,  4  suppressive  0 ),  suppressive  on "B" responder c e l l s i n a B vs A MLR;  a c t i v i t y o f "A" TsF a c t i n g on "B" r e s p o n d e r c e l l s  i n a B vs A a l l o g e n e i c MLR.  A l l MLR a r e one-way; i n each  each column f r a c t i o n e l u t e d from B16G-A8 ( d i a l y z e d a g a i n s t  c a 3 e  100 j i l o f  DME) were added.  33  Table I Effect of syngeneic HTsF + B16G on a human MLR and on response to mitogen  Concentration of HTsF added (per ml)  Responders + Stimulators (cpm/2-ml culture ) a  Responders + Stimulators + 100 yg Irrelevant MAb  0 yg  16,584 +  600  3 yg  5,016 +  256  9,864 + 1,512  3,814 + 504  10 yg  1,409 +  72  3,801 + 1,345  1,793 + 237  30 yg  939 +  48  2,382 +  122  100 yg  580 +  30  2,365 +  64  1,095 +  33  612 +  18  Responders only Stimulators only Responders + PHA  99,386 + 6,000  Responders + PHA + 30 yg TsF  92,239 + 3,115  a  Responders + Stimulators + 100 yg B16G  b  968 + 128  Each data point represents the average of four r e p l i c a t e cultures.  ^Standard deviations are also included.  34  Figure 6. SDS-PAGE analysis of polyclonal mouse TsF. Lane 1, DBA/2 splenocyte membrane extracts purified over B16G-4B columns; Lane 2, 1/20 dilution of starting material prior to immunosorption; Lane 3, spleen extracts "purified" over an irrelevant monoclonal column; Lane 4, 1/20 dilution of B16G column fall-through material. extract (as assessed by ODgso  For lanes 1 and 3, the same amount of spleen u n  ^ ) t s  w  a  s  a  P P l i i to the columns. For e <  purposes of SDS-PAGE analysis only the peak ELISA reactive fractions are used (Fraction 4, Plgure 1).  35  In order to establish which materials seen on the gels reacted with B16G, a gel was sectioned horizontally into strips spanning 10-15 kD; the proteins from each strip were eluted into low ionic strength buffer and tested for reactivity with B16G in the ELISA.  The results (Figure 7) show  clearly that major reactivity was observed in the material eluting in the 40-50 kD range which coincided with the major bands observed on the gels. There was also minimal reactivity above background with B16G in the material eluting in the 90-100 kD range; this may represent the native, undissociated molecule of TsF. Characterization of the human derived Bl6G-reactive material was also carried out on PAGE gels. shown in Figure 8A and 8b.  Representative  runs of such preparations are  Human tonsillar tissue extracts yielded  relatively pure material upon elution from B16G columns.  In tracks B and  C (Fig. 8B), two different ELISA reactive fractions eluted from B16G immunoadsorbents from tonsil preparations are shown. In both cases, a band of approximately 90 kD is seen as well as stronger bands at between 45-50 kD.  In some gels (Figure 8A), the 90 kD band was not visible  indicating the apparent instability of this moiety.  The associated 25 kD  band can be seen in this gel, especially in lane B. Further experiments were carried out to determine the native molecular weight of the B16G reactive material.  For this purpose, TsF  eluted from B16G columns was cofractionated with BSA and myoglobin using a Sephadex G-150 column.  The column was run at 4°C, and 3.0 ml fractions  were collected. Each fraction was lyophilized, and aliquots were tested either in the ELISA or run on SDS-PAGE. ELISA results of the G-150 column  36  Figure 7.  ELISA profile of material recovered from SDS-PAGE gels to which  pooled B16G eluates were applied. Lanes from a reducing SDS-PAGE gel over which pooled B16G column eluates and pooled irrelevant MAb-4B column eluates were run were sectioned into horizontal strips, each spanning approximately 10,000 Daltons MW.  Materials in each strip were eluted into  buffer and analyzed (in quadruplicate) in the ELISA assay for TsF (B16G reactivity). shown.  Approximately molecular weights spanned by each strip are  ( • ), ELISA reactivity recovered from B16G-4B eluate lane  (Fig. 6, lane 1); ( • ), ELISA reactivity recovered from irrelevant MAb eluate lane (Fig. 6, lane 3). times.  This type of experiment was repeated three  A .  jL;.  B  C  D _ , „ . _ J .  Figure 8A. SDS-PAGE analysis of human tonsillar extracts purified over B16G-4B. Lane A, low MW standards 5 pg ovalbumin (43 kD) and chymotrypainogen A (25 kD); Lane B and C, HTsF eluted from B16G columns; Lane D eluates of tonsillar extracts from irrelevant (anti-human lung antigen) columns.  For purposes of gel analysis only peak ELISA-reactive  fractions from the B16G column were used (Fraction 4, 5, Fig. 4).  A  F i g u r e 8B. ovalbumin,  B  C  D  E  SDS-PAGE g e l a n a l y s i s o f human p o l y c l o n a l T s F . ( m o l e c u l a r weight 4 3 , 0 0 0 k D ) ;  Lane A ,  Lane B and C, t o n s i l l a r c e l l  membrane e x t r a c t s p u r i f i e d over B16G-4B, d i a l y s e d a g a i n s t d i s t i l l e d and l y o p h i l i z e d p r i o r t o g e l a n a l y s i s ; Lane D,  t o n s i l l a r e x t r a c t s passed  over and e l u t e d from i r r e l e v a n t monoclonal columns; Lane E, e x t r a c t p r i o r t o immunosorption,  H^O  tonsil  cell  the same amount o f t o n s i l e x t r a c t was  a p p l i e d t o both B16G and i r r e l e v a n t columns.  39  eluates for murine TsF are shown in Figure 9, in which i t can be seen that the major Bl6G-reactive component eluted in the range of 80 kD molecular weight (fractions 3-5).  When these same samples were run on SDS-PAGE, i t  was apparent that the 80 kD material was composed of subunits which ran at 45 kD and 25 kD molecular weight.  The gel signature thus obtained  was  comparable to that obtained by PAGE analysis of material eluted from the B16G column (not shown). In order to establish that the 80 kD material eluting from Sephadex G150 columns had suppressive biological activity, individual fractions were tested at a 1:8 dilution for their ability to suppress the  MLR.  Results are also shown in Figure 9 in which i t can be seen that the suppressive activity in the fractions coincides with material eluting in the 80 kD range and to a smaller extent with material eluting in the 45 kD range. It therefore appears that the material which reacts with B16G and has immunosuppressive properties occurs in i t s native form as a molecule with a molecular weight in the range of 80 kD, which under reducing conditions may run on gels as subunits with molecular weights of 45 kD and 25 kD. In order to determine the approximate molecular weight of the human Bl6G-reactive material, eluted materials were passed over a Sephadex G150 column, and individual fractions following the void volume were tested in ELISA for Bl6G-reactivity.  It can be seen in Figure 10 that most of the  reactive material elutes in fractions indicating a molecular weight of 80 kD ± 10 kD.  However, a lesser but reproducibly reactive fraction was  40  F i g u r e 9. G150  ELISA p r o f i l e of B16G-4B column e l u a t e s f r a c t i o n a t e d  Sephadex column.  P o o l e d e l u a t e s from 16 DBA  presensitized  e q u i v a l e n t s passed o v e r the B16G-4B columns were a p p l i e d m o l e c u l a r sieve  i n 10% g l y c e r o l .  100 j i l a l i q u o t s d i l u t e d 1:2 plates  in quadruplicate f o r analysis.  f r a c t i o n s e l u t i n g from a G-150 i n the MLR 100%  from column f r a c t i o n s . l e s s than  4 Q 5  );  column.  a t a f i n a l d i l u t i o n of 1:8.  values f o r the MLR 10%.  calculated The  G150  3 ml f r a c t i o n s were c o l l e c t e d a t  i n c a r b o n a t e b u f f e r were a p p l i e d  e l u t i o n p r o f i l e , (ELISA 0 D  spleen  t o the  4°C.  t o ELISA  Approximate m o l e c u l a r w e i g h t  spanned by each 3 ml f r a c t i o n i s shown below each f r a c t i o n #. G-150  over a  ( 0—0  ),  ( a ), s u p p r e s s i v e a c t i v i t y of Individual  f r a c t i o n s were  tested  P e r c e n t i n h i b i t i o n i s based  from c u l t u r e s  lacking material  on  added  standard e r r o r o f the mean f o r each p o i n t  was  41  2  F i g u r e 10.  4  8  10  13  U 16 MICTION'  18  20 •  ELISA a c t i v i t y p r o f i l e o f p u r i f i e d  G-150 Sephadex. B16G-4B; m.w.  4  HTsF f r a c t i o n a t e d o v e r  ( t — - 0 ) , ELISA p r o f i l e o f 100 u l HTsF p u r i f i e d o v e r  c o r r e s p o n d i n g t o each ELISA peak i s shown.  Column  was  c a l i b r a t e d w i t h b l u e d e x t r a n (150,000 d a l t o n s ) , BSA (68,000 d a l t o n s ) , and myoglobin (20,000 d a l t o n s ) , ( 0  0 ).  42  detectable with a molecular weight in the region of 40-50 kD.  The  properties of the human tissue derived material, thus, are essentially the same as those described above for murine TsF. In an attempt to demonstrate the efficacy of the B16G molecule as a probe to evaluate relative levels of suppression in pathological conditions, a study was undertaken to establish whether a generalized suppressor c e l l defect occurs in MRL-lpr mice (autoimmune murine SLE model).  Since B16G had been shown to react with TsF in the spleens of  normal mice and with polyclonal whole human TsF isolated from tonsillar tissue, we used this monoclonal antibody (which may display specificity to a constant region epitope common to both human and murine T suppressor factor) as a probe to evaluate the level of functional suppressor factor present in MRL/lpr mice when compared to a MHC compatible strain of mouse, CBA.  TsF present in the spleens of these animals was purified by B16G-4B  immunoadsorbence, and tested for suppressor activity in an allogeneic MLR, as described in the methods for DBA polyclonal TsF.  No difference was  detected in functional TsF yield or activity between the strains suggesting that there is no generalized suppressor defect in MRL-lpr mice.  Figure 11 demonstrates that when MRL/lpr activity was assessed by  the ability to suppress a unidirectional allogeneic mixed lymphocyte reaction, (TsF's isolated from both CBA and MRL-lpr splenocytes were added at concentrations of 2 yg, 1 yg, 0.5 yg/ml) both T suppressor factors could effectively suppress the T lymphocyte response from CBA mice (H-2 ) to x-irradiated C57B1/6 (H-2 ) stimulator cells. k  b  It is  interesting to note that the CBA TsF was less effective in  43  CBA vs. B6j }*CBATsF Ir  CBA«.B6frr.)+MRL TsF B6vs.CBA(, )»CBATsF rr  a z z  o 3  9 Oul  25ul CBA TsF  50ul  Oul  2Sul MRL TsF  50ul  O ul  25ul CBA TsF  50ul  Figure 11. Suppressive activity of MRL and CBA B16G eluates. Bl6G-eluted fractions of each spleen extract which scored highly in the ELISA analysis were tested for bioactivity in a unidirectional allogeneic MLR.  Both CBA  and MRL TsF's (H-2**) were added (in separate experiments) to CBA responders.  5 day allogeneic response directed to irradiated C57B1/6  (H-2b ) target cells was measured by 3 H-Tdr incorporation in octuplicate.  Cells were precipitated onto glass f i l t e r s (Whatman) with  TCA in a standard MASH harvester. Samples were read for tritium incorporation on a scintillation counter. 3  Stimulation index refers to the  ratio of H-Tdr incorporation in reactive cells over unstimulated controls. Columns 1, 2, 3 suppression of CBA vs. irradiated B6 cells (irr) MLR by 0, 25 and 50 y l of CBA TsF; Columns 4, 5, 6, suppression of CBA vs. B6 (irr) MLR by 0, 25 and 50 y l of MRL lpr/lpr TsF; Columns 7, 8, 9, suppression of a B6 vs CBA (irr) MLR by CBA TsF. was performed 4 times.  This class of experiment  44  suppressing the reciprocal MLR (C57B1/6 anti-CBA), consistent with the element of genetic restriction present in the effector function of this factor.  The factors were not themselves toxic as their addition failed to  influence background proliferation (data not shown).  DISCUSSION  In this chapter, i t has been demonstrated that a MAb originally directed to DBA/2 anti-P815 murine TsF apparently reacts with, and can be used to purify, analogous material from both murine spleens and human lymphoid tissue.  Clearly, the credibility of such a study hinges  primarily on the pedigree of B16G-MAb i t s e l f .  This MAb had been shown to  abrogate suppression in several independent DBA/2 tumor systems (112), and was demonstrated by panning and tumor protection studies to recognize a determinant common to a l l DBA/2 TsFs.  Since the homology between many  murine and human immunological proteins (MHC, Ab, complement receptors) i s high, i t was felt possible that the epitope recognized by B16G in murine TsF might be repeated in HTsF. This apparently is the case.  As in the  murine system, B16G columns will absorb detectable levels of protein from human lymphoid tissue, whereas no yields at a l l are obtained from columns containing irrelevant MAb of the same subclass (even those directed to other human antigens). Material isolated from tonsil or spleen lysates over B16G is ELISA-reactive; most importantly, the reactivity of eluted material with B16G correlates directly with i t s immunosuppressive function  45  i n the MLR.  H i g h l y s u p p r e s s i v e HTsF i s o l a t e s from one donor w i l l not a c t  as e f f e c t i v e l y w i t h another donor's c e l l s , that t h e e f f e c t observed  o f d i f f e r i n g HLA, i n d i c a t i n g  i s not n o n - s p e c i f i c , o r due t o lymphotoxins and  that i t may be M H C - r e s t r i c t e d .  This result  i s i n agreement w i t h o t h e r T s F  r e s e a r c h e r s ' r e s u l t s , n o t a b l y that o f Fresno e t a l .  ( 1 1 3 ) ,  who have  e f f e c t i v e l y demonstrated I - J r e s t r i c t i o n i n T s F d i r e c t e d to g l y c o p h o r i n (sheep  RBC).  The same group, amongst o t h e r s , have demonstrated t h a t the  p h y s i c a l c h a r a c t e r i s t i c s o f t h e i r molecule  - a 4 5 kD s u p p r e s s i v e c h a i n and  2 5 kD a n t i g e n b i n d i n g c h a i n - p a r a l l e l s r e s u l t s observed murine and human systems.  by us i n both the  Murine TsF's and HTsFs i s o l a t e d by B 1 6 G  a b s o r p t i o n can be r e s o l v e d by SDS PAGE i n t o a 4 5 kD band ( o f t e n a p p e a r i n g as a dimer o f two narrowly associated protein.  separated i n d i v i d u a l p r o t e i n bands) and a 2 5 kD  A band running a t a p p r o x i m a t e l y  (HTsF) was a l s o f r e q u e n t l y observed, molecule,  7 0 kD (MTsF) o r 9 0 kD  and may r e p r e s e n t t h e i n t a c t T s F  the m o l e c u l a r weight o f which has been demonstrated as  approximately  8 0 kD ± 1 0 kD by Sephadex G 1 5 0 column f r a c t i o n a t i o n .  These o b s e r v a t i o n s a l s o e s s e n t i a l l y agree w i t h t h e work o f Fresno (113)  which i n d i c a t e s t h a t 4 5 kD and 2 5 kD elements compose t h e T s F  molecule,  and a r e a l s o i n agreement w i t h t h e s t r u c t u r e o f a KLH TsFs  d e s c r i b e d by T a n i g u c h i and Tada  ( 1 1 4 , 1 1 5 ) .  More r e c e n t l y , these o b s e r v a t i o n s were expanded t o o t h e r s t r a i n s o f mice. was  T h i s technique  o f p o l y c l o n a l T s F p u r i f i c a t i o n over a.Bl6G column  used i n o r d e r t o compare the r e l a t i v e t o t a l l e v e l s o f T s F c o n t a i n e d i n It  the spleens o f normal C B A ( H - 2 ) and MLR/lpr autoimmune mice (murine lupus model).  M a t e r i a l o b t a i n e d from each had t h e same c h a r a c t e r i s t i c s as  46  those described above, appearing as a 45 kD band with an associated 25 kD subunit, and specifically reacting in ELISA with B16G and a rabbit anti-TsP antiserum.  CBA and MRL B16G eluates both were suppressive in  allogeneic MLRs (Figure 11).  The phenomenon of MHC restriction was also  observable in these TsF preparations in that, for example, CBA TsF will inhibit only the allogeneic response of CBA (H-2 ) cells in the MLR; the ability of C57/B16 cells to respond is unimpaired.  These observations  indicate that the suppressive effect observed is not due to non-specific causes (e.g. lymphotoxins), and are similar to those obtained in the human system. In summary, these observations indicate: 1.  B16G can be a useful probe for isolating TsC and TsF from human lymphoid cells and at least 4 murine strains.  2.  The antibody clearly recognizes a constant epitope on the TsF (TsC receptor?) molecule common to mouse and man.  3-  B16G immunoadsorbents can be used for large-scale purification of TsF molecules directed towards any antigen or idiotype for analysis biochemically.  4. B16G can be useful in the assessment of relative levels of suppression to specific antigens (e.g. MRL/lpr vs. CBA mice), which is important information in a variety of human conditions.  47  Chapter Summary  We have shown that a murine monoclonal antibody, B16G, recognizes a constant region determinant of a T c e l l suppressor factor (TsF) in DBA/2 mice.  The murine TsF molecule recognized by B16G was shown to be a  protein molecule with a native molecular weight in the region of 80 kD ± 10 kD. We also show that B16G also reacts with a similar molecule derived from human lymphoid tissue.  Yields of about 10 pg o  could be obtained from the solubilized membranes of about 10  tonsillar  cells by elution of adsorbed materials from B16G immunoadsorbent columns. In both the murine and human system, TsF thus derived was capable of suppressing the mixed leukocyte reaction (MLR) of homologous responder T lymphocytes.  However, this same material was much less suppressive  across the HLA or H-2 barrier, that i s , when allogeneic effector cells were used in the MLR.  Preliminary characterization of both the HTsF and  murine TsF showed i t to have a native molecular weight of 80 to 90 kD, and to be readily broken down to yield subunits with molecular weights in the region of 45 - 50 kD and approximately 25 kD. These observations provide evidence for the conserved nature of genes encoding TsFs and correlate with observations of other investigators on the considerable homology between genes encoding the murine and human T c e l l receptor (116,117).  48  Chapter II  Isolation and Characterization of a Tumor-Specific T-suppressor Factor  (TsF) from a T-Cell Hybridoma INTRODUCTION  In the past two years, a number of approaches including the use of monoclonal antibodies (MAb) directed to T cell surface specific components of cloned T c e l l lines or T cell hybridomas, and cloning of genes rearranged only in T cell lines - have led investigators closer to defining the antigen-recognizing molecules located on the surface of T cells. Using cloned human CTL lines, and MAb directed specifically to unique epitopes on these lines, Reinherz and coworkers (116) have identified a heterodimer of 49 and 43 kD molecular weight as the T cell receptor on these cells.  This molecule is associated with but not covalently bound to  the T3 molecule, common to a l l human T cells.  In the murine system,  Kappler et a l . (117) have demonstrated antigen-binding molecules on T cell hybridomas to be heterodimeric structures in which the subunit molecular weights were 43 and 40 kD.  It is possible that variations in molecular  weights may be attributable to differences in the number of N-linked glycosylated groups, 5 in the murine molecule, and probably 2 in the human. In more recent studies, Mak and coworkers (118), succeeded in cloning a gene from the human T cell line MOLT 3, which is rearranged in the T  49  c e l l line but not in control B cell lines.  The gene product was assumed  to have a molecular weight in the region of 35 kD, and projected amino acid sequences showed homology with Ig V and C gene products.  Similarly,  Hedrick and Davis (119) have isolated analogous clones from murine T cells using a slightly different approach.  It has since been conclusively  demonstrated that these genes encode the antigen binding region of the T cell receptor for THC's and CTL's. A number of questions remain to be answered.  The a and B genes  from Ts c e l l lines (which do not rearrange in this region) have yet to be assigned a role as the antigen-binding component of the Ts cell receptor. Also, while there is good evidence that the heterodimer identified with MAb constitutes an antigen binding site on a variety of differentiated T cells, the connection between i t and soluble factors secreted by helper or suppressor T cells has yet to be established. In this laboratory, the monoclonal antibody B16G had been demonstrated to react with the TsF's which specifically inhibited the development of cytotoxic T cells to the syngeneic P815 DBA/2 mastocytoma and which could be purified by absorption and elution from columns containing P815 membrane extracts. B16G was subsequently shown to prolong survival of mice injected with unrelated tumor cell lines.  These  observations led to the assumption that B16G was reacting with a constant region on a TsF secreted by regulatory T cells in DBA/2 mice.  In further  studies, i t was found that B16G could enhance MLR reactivity in splenocytes of DBA/2 mice injected with B16G two days prior to sacrifice, or when added to MLR cultures, along with extracts from D B A / 2  50  splenocytes.  It was suggested that B16G was recognizing an epitope from a  constant region of a regulatory molecule secreted by, and present on the surface of, DBA/2 Ts lymphocytes (120,121,122). The study presented here involved the use of B16G as a probe to isolate T c e l l hybridomas secreting TsF. The DBA/2 mice used in this study were injected 4 days prior to sacrifice with a membrane extract of P815 mastocytoma cells, a protocol known to enhance development of TsC in this system. Supernatants from fusion products derived from P815-primed DBA/2 thymocytes and the c e l l line BW5147 were screened for reactivity with B16G in ELISA.  A fusion product, the A10 T cell hybridoma, i s  described here, in which i t is shown that this hybridoma secretes a product which reacts specifically with both B16G and P815, and which in vivo, specifically inhibits inherent immunity to the growth of P815 in DBA/2 mice.  Preliminary biochemical characterization of this  tumor-specific TsF is also shown.  MATERIALS AND METHODS  Experimental animals.  DBA/2 female mice between 6 to 8 wk of age were  used for a l l in vivo assays of TsF.  CBAxBALB/cF^ mice were used to grow  T c e l l hybridomas as an ascities tumor.  These were injected on day -5  intraperitoneally (i.p.) with 0.5 ml pristane, irradiated on day -1 with 400 rads, and injected on day 0 with at least 10^ viable hybridoma cells i.p. in phosphate-buffered saline (PBS). B10.BR splenocytes from mice between 6 and 8 weeks of age were used as targets in the MLR. A l l animals  51  were raised In the Microbiology Department animal f a c i l i t y at this University.  Cell lines.  The L1210 leukaemia, P815 mastocytoma, and the M-l  rhabdosarcoma of DBA/2J mice used in this study have been maintained in this laboratory for over 10 years.  Membrane extracts of P815 cells used  for immunization were obtained by sonication and high-speed centrifugation of P815 ascites tumor cells as described (111,112).  The fusion partner  BW5147 was obtained from ATCC, and has been maintained both by passage in culture or by storage in 10% dimethylsulfuxide-frozen cultures at liquid nitrogen in this laboratory for the past 5 years.  Testing of BW, and the  other lines, for mycoplasma is carried out on a regular basis.  Fusion.  DBA/2 mice were injected i.p. on day -5 with 200 v>g of P815  membrane extract. On day 0, three such mice were sacrificed and their thymuses were removed. Thymocytes were made into a single c e l l suspension by squeezing through nylon mesh and repeated passages through a 26-gauge needle.  Cells were then washed twice by centrifugation in  3terile  PBS,  and were counted for viable cell numbers by using trypan blue exclusion. The BW5147 cells to be fused were grown to log phase in Dulbecco's minimal essential medium (DMEM) and 15% fetal calf serum (FCS).  Usually,  7 about 10 BW5147 cells were required. Cells were grown to no greater density than 5 x 10^ cells/100 ml media. BW5147 and immune splenocytes at a 2:1 ratio were mixed and were centrlfuged together (1800 rpm, 10 min)  52  into a pellet; 10% polyethylene glycol (m.w. 3500; Serva Feinbiochemica) was added slowly over a 2-min period. Fusion products were plated out on CBA x BALB/cF^ thymocyte feeder 5  layers (2 x 10  cells/well) in 96-well tissue culture plates (Falcon)  and were grown in DME, (20% FCS, and hypoxanthine-aminopterin-thymidine; HAT).  Throughout a l l subsequent growth and cloning steps, the hybrids  were maintained continuously in this medium and with the feeder layer. Selection procedures.  Hybrids which were growing well were transferred on  day +20 to 2.0 ml-well tissue culture trays (Linbro) for aditional study. Again, they were maintained continuously in HAT medium and feeder layer. Culture supernatants (1.0 ml) from each well were collected and assayed for TsF by immunoadsorption from columns to which the anti-TsF monoclonal B16G had been linked to Sepharose 4B.  Eluates (eluted by 0.1 N HC1) were  neutralized in 7.0 M Tris-buffer and were applied to Immulon-2 ELISA plates in carbonate buffer (pH 8.6) (1/1 dilution) for analysis in the ELISA.  ELISA.  Plates coated with material eluted from the B16G columns were  f i r s t blocked by using rabbit anti-mouse Ig (RaMIg) to eliminate any background effects due to B16G molecules washed off the column during the elution.  RaMIg was added at a concentration of 200 pg per well in PBS  for 1 hr at room temperature. Chapter I.  Other procedures were as described in  53  Figure 12. Flow diagram illustrating the method used to isolate the A10 hybridoma.  Primed DBA/2 spleens were fused to BW5147 cells and their  supernatants screened for Bl6G-reactive material by passage over B16G-4B columns.  Eluted materials were coated on ELISA plates for analysis.  54  Hybrids scoring positively in these tests were transferred to 10 ml culture flasks (Falcon) for additional study, and again were continuously maintained in HAT medium, 20% FCS, and feeder layer.  The remaining  hybrids were discarded, though some were retained to serve as negative controls.  Hybrid supernatants scoring positive in the ELISA were also  tested in a similar manner after elution from 4B columns to which inactive mouse antibodies of the same subclass as B16G had been linked.  These  served as controls to test against irrelevant binding of hybrid  proteins  to Sepharose 4B or to B16G.  Immunoadsorbents.  To affinity purify TsF from A10 ascites f l u i d , an  immunoadsorbent column consisting of Sepharose 4B to which the ctTsF MAb B16G had been linked by CNBr was used.  Twenty m i l l i l i t e r s of fresh  ascites fluid was applied to a 10-ml B16G-4B column (maintained at 4°C; non-adherent material was washed away by using sterile PBS at 4°C until no detectable absorbence at 280nm remained in the column wash.  Elution of  Bl6G-adherent material was then carried out by using 0.1 N HCL; eluted material was immediately neutralized with 150 y l 1.5 M Tris-HCl, pH 7.0.  Control columns consisting of Sepharose 4B only or Sepharose 4B  linked to irrelevant monoclonals of the same subclass as B16G were also used.  Specificities of the "irrelevant" MAb used as control  immunoadsorbents were directed to ferredoxin and a protein associated with the membrane of the human lung cancer cell line A-549 (IgM class). anti-ferredoxin antibodies were of subclass IgG  The  , the same as B16G.  55  To teat the antigen specificity of A10 TsF, a column consisting of Sepharose 4B to which P815 membrane extracts had been bound by CnBr was used.  Again, A10 or control ascites were applied, and P815-adherent  material was eluted with 0.1 N HC1.  Eluates were neutralized and applied  to ELISA plates to test for B 1 6 G reactivity.  Controls in these  experiments included columns to which M-l tumor c e l l extracts or L1210 tumor cell extracts had been conjugated to Sepharose 4B beads.  MLR. Supernatants from A10 c e l l cultures and from appropriate controls were passed over a B16G immunoadsorbent, and the eluate was tested for general activity in the MLR. Splenocytes from DBA/2 mice were cultured at 5  a concentration of 3 x 10 cells/well with various concentrations of A10 or control B16G eluates in RPMI 1640 containing 5 x 10~ M 5  2-mercaptoethanol and 10% FCS in Falcon microtiter wells (no. 3072). Irradiated splenocytes (1000 rad) from B10.BR mice were co-cultured as 5  stimulators at a concentration of 3 x 10 cells/well in a total volume of 200 |il.  After 72 hr incubation at 37°C in a humidified  incubator  at 5% C0 , 50 pi of [ ]H-thymidine (20 pCi/ml) in RPMI 1640 was 3  2  added to a l l cultures.  Cells were harvested 18 h later on a MASH  harvester and were counted for 1 min on a Packard scintillation counter (TRI-CARB Model 4550).  Controls consisted of 3 x 10 DBA/2 cells 5  cultured alone with the various concentrations of column eluates. A l l test results were averaged from six individual replicates.  56  In vivo analysis of TsF.  To assess the effect of A10 TsF in vivo, A10  cells were f i r s t grown as an ascitic tumor in CBA x BALB/cF.^ mice. On day +14, mice were tapped for ascites by using an 18-gauge needle; the ascites was diluted 1/2 in PBS (sterile) and was purified over and eluted from a large (10 ml) B16G-4B column with 0.1 N HC1. Peak fractions (as determined by their 280 nm absorbence) were immediately neutralized in 1.0 M Tris buffer, and were dialyzed overnight against PBS at 4°C.  The  purified TsF was then injected intravenously (i.v.) into DBA/2 mice that 4 had received an injection of 1x10 viable P815 cells subcutaneously in the flank the same day (or, in some experiments, 2 days previously). Dialyzed B16G eluates from the ascites of non-TsF-producing hybrids  (e.g.,  G10), or the BW5147 parent line, both injected i.v., served as controls. To determine the native M.W. of the TsF molecule, a 50% ammonimum sulphate cut of A10 ascites was prepared, dialyzed against PBS, and run over a G-150 Sephadex column.  Fractions scoring highest in the ELISA (in  the region of M.W. 80,000) were pooled for injection into DBA/2 mice concurrently with the P815 tumor, as described above.  The course of P815  growth in these animals was monitored by daily measurement of tumor area with calipers until the f i r s t deaths in each group occurred.  Eight mice  were used in each group; the results were subjected to standard statistical (Student's t-test) analysis. Other tumor lines were also tested as controls to test A10 TsF specificity for P815. from DBA/2 mice. described above.  These include L1210 leukaemia and M-l rhabdosarcoma  These tumors were grown in A10- or GlO-treated mice as 3 The tumor dose for L1210 was 3 x 10 cells, and 1 x  57  10  for M - l .  The positive controls of P815-injected mice were tested  concurrently.  Statistical analyses.  Tumor measurements on experimental animals were  made daily, and area measurements for each group were compared with PBS control values by Student's t-test.  Differences were considered to be  significant at p values of <0.05.  SDS-PAGE. A10 material eluted from the B16G column was resolved on SDS-PAGE. 10% polyacrylamide gels (120,121) were used.  Samples to be run  were neutralized, frozen, and lyophilized overnight at -50°C. Samples were stored frozen at -70°C; just before application to the gel, samples were taken up in 60 jil d i s t i l l e d HgO and 40 y l reduction mix (2.3% SDS, 15% glycerol, 1% dithiothreitol (DTT), 12.5% Tris, pH 6.8, and bromphenol dye, 0.1%).  Samples to be run on nonreducing gels were  dissolved in 60 ul d i s t i l l e d HgO and sample buffer (as above) without DTT. Gels were silver stained according to standard methods (9).  In some  cases, bands were eluted from the gel for assay in the ELISA as in Chapter I. Silver-stained gels for analysis were f i r s t destained in Kodak Rapid Fix (15) and 50% methanol, and were sectioned into horizontal strips spanning 10,000 kD M . W .  Proteins were eluted from these strips in 1.0 ml  PBS overnight at 4°C. Eluted material was coated on ELISA plates (200 ul/well) in carbonate buffer, and the ELISA's were run as described above.  58  CTL  Mlcroa33ay.  Virgin or panned DBA/2 spleen  cell3  were plated in  doubling dilutions in V bottom plates (Linbro #76-023-05) from 10 to 6  5  1.25 x 10 cells in 100 yl aliquots per well in replicates of 8. P815 cells were obtained from ascites fluid of DBA/2 mice, washed 3 times and incubated at 2 to 6 x 10^ cells/ml with 50 yg/ml mitomycin C in _5 complete RPMI (RPMI, 10 mM Hepes, 5 x 10 M 2-mercaptoethanol, 10% PCS, penicillin/streptomycin) at 37°C and 4% C0 for 1 h. P815 cells were 2  then washed 3 times, resuspended in complete RPMI and 100 y l of media containing 2.5 x 10 cells was added to each well containing spleen cells.  5 days later cells were resuspended and 100 y l from a l l wells  was transferred to U bottom plates (Linbro #76-24-205). Fresh P815 cells 51 6 were labeled for 1-1/2 h with Cr at 0.2 mCi/2xl0 cells. The cells were washed, incubated at 37°C and 5% CO^ in complete RPMI for 3 to 4 h.  The labeled P815 cells were washed 3 times, resuspended in complete 4  RPMI and 100 yl containing of 10 cells was added to a l l wells including 2 rows of 8 replicates of labeled P815 alone for calculation of spontaneous and maximum chromium release.  The plates were centrifuged at  100 rpm for 5 min and then incubated at 37°C and 5% CO^. After 18 h plates were again centrifuged and 100 y l of supernatant was removed from a l l wells except from rows of P815 cells alone in which 100 yl of cell suspension was removed to calculate maximum chromium release.  Samples  were counted in a Picker Pace 1 gamma counter. % specific lysis was calculated as follows: % specific lysis =  sample cpm - spontaneous cpm x 100 maximum cpm - spontaneous cpm  59  RESULTS  Selection of T Cell Hybridomas.  Fusion of P815-sensitized DBA/2  splenocytes with BW5147 cells was carried out as described.  Microtiter  wells were scrutinized microscopically for the presence of fusion products.  Those wells containing hybridomas were expanded to 2.0 ml wells  and allowed to grow up.  Since we had previously shown that the monoclonal  antibody B16G appeared to bind to a constant region of DBA/2 TsF (120,121, Chapter I), B16G-immunoadsorbent columns were used to identify T c e l l hybridoma supernatants secreting putative TsF.  From each of the 2.0  ml cultures, 1.0 ml of culture supernatant was passed over small columns (approximately 500 yl) of Sepharose 4B conjugated B16G.  The reason this  method was chosen rather than directly coating ELISA plates was due to the fact that FCS in the medim could coat the plate and block B16G binding. This method removes most contaminating proteins other than TsF.  Material  eluted with 0.1 N HC1 was neutralized, diluted 1:1 with ELISA bicarbonate coating buffer and used to coat ELISA plates. tested for ELISA reactivity with B16G. products were tested.  This material was then  In this manner, 3,504 fusion  Of a l l these, 13 B16G-reactive hybridomas were  recovered, and of these, the A10 hybridoma, had the strongest reactivity. It was thus selected for further analysis.  Three other positive hybrids  were frozen down and stored for some future study.  The A10 hybridoma was  cloned, tested, and expanded, and larger volumes of culture supernatant were tested for B16G reactivity after immunoadsorbence.  Figure 13  demonstrates the B16G reactivity recovered from 10 ml of A10 culture  fMCtlCM'  F i g u r e 13.  E l u t i o n p r o f i l e s o f AlO and G10 c u l t u r e s u p e r n a t a n t s  immunoadsorbent columns.  Bottom l i n e s r e p r e s e n t r e s u l t s  s u p e r n a t a n t s , from which no ° was o b t a i n e d .  n 2 8 0  ( 0-  from B16G  o b t a i n e d from G10  0 ) o r ELISA a c t i v i t y  ( 0---0  No ELISA a c t i v i t y was o b t a i n e d from 3,500 h y b r i d s t e s t e d  s i m i l a r l y , o r from t h e BW5147 parent l i n e s u p e r n a t a n t  ( n o t shown).  )  61  supernatant eluting from a B16G column in comparison to an analogous preparation from a negative T cell hybridoma, G10. It can be seen that the A10 supernatants contain significant levels of Bl6G-reactive material whereas the control does not.  Similarly,  3,500 other hybrids and the  BW5147 parent line also gave no B16G activity in this assay. It was found that A10 and control negative hybridomas could be grown as ascites tumors in CBAxBalb/c F l irradiated mice.  When ascites fluid  from these hybridomas was passed over B16G immunoadsorbents and eluted, detectable amounts of 280nm-absorbing material was eluted from A10 ascites whereas negligible amounts were detected with G10 (or other Bl6G-negative hybridomas tested i n this way).  The amount of protein eluting from these  columns correlated directly with the intensity of reactivity of eluted fractions with B16G in the ELISA (as in Fig. 13) relative to control (G10) ascites.  When A10 ascites were passed over an irrelevant immunoadsorbent,  essentially no 280 nm absorbing material was eluted and no ELISA reactivity in equivalent fractions was observed (data not shown). The A10 hybridoma, i f the Bl6G-reactive material i t produces constitutes a variety of antigen-specific TsF, should demonstrate antigen specificity.  Since animals from which A10 was derived had been primed  with P815 membrane extracts, attempts were made to determine i f the A10 ascites would bind specifically to a P815 membrane extract immunoadsorbent.  Consequently, A10 ascites were passed over a P815  column, eluted, and tested for reactivity with B16G in the ELISA.  It was  found that Bl6G-reactive material was eluted from such columns when A10  62  ascites was passed over them whereas no such material was eluted from the P815 column over which G10 or BW ascites had been passed (Fig. 14 and 15). It was recognized that the ability of A10 ascites fluid to bind to P815 columns did not constitute a complete test for the specificity and biological activity of this material.  Experiments were therefore  undertaken to determine i f A10 ascites fluid could influence the growth of P815 in DBA/2 mice.  In the f i r s t experiment, ascites fluid from A10 and  G10 were precipitated with 50% saturated (NH^JgSO^, dialyzed against PBS, and injected i.v. at a concentration of 25.0 yg into DBA/2 mice two 3  days prior to challenge with 4 x 10 P815 cells administered subcutaneously (s.c.) Mice were examined daily for appearance of tumors and tumor size was measured daily with calipers.  The results (Fig. 16A)  showed that A10 administration enhanced significantly the growth of P815 tumors whereas G10 material did not alter tumor growth in comparison to the PBS control; survival times of these mice were also significantly reduced (Fig. 16B). A second series of experiments were run using Bl6G-absorbed A10 and G10 ascites.  Ascites materials were eluted from B16G columns, dialyzed  overnight against PBS and injected, (in equivalent volumes) i.v. into 4 DBA/2 mice which had been injected 30 min previously s.c. with 1 x 10 P815 cells.  The results (Fig. 17) again show that Bl6G-reactive material  affinity purified from A10 ascites enhanced P815 growth as opposed to the PBS control.  Additionally, A10 material purified over P815-4B columns had  a similar effect.  No suppressor effects were noted from A10 "purified"  over irrelevant columns (not shown).  I i  • AIO<MrM-l  F i g u r e 14.  E l u t i o n p r o f i l e s o f AlO and BW5147 a s c i t e s passed o v e r  Ag-4B immunoadsorbent column:  ( •  • ), OD  P815  o b t a i n e d from 20 ml AlO 280  (5 mg/ml) passed o v e r P815-4B; ( •••»«•-•• ),  0 0  2BO  BW5147 a s c i t e s (5 mg/ml) e q u i v a l e n t l y t r e a t e d . the column i n t h i s way  O  B  T  A  I  N  E  from 20 ml  D  AlO m a t e r i a l e l u t e d  from  i s both h i g h l y ELISA r e a c t i v e and i d e n t i c a l i n  appearance on SOS-PAGE t o AlO p u r i f i e d o v e r B16G-4B; ( t a s c i t e s passed o v e r an M—1-4B a d s o r b e n t  column.  1  • ) , AlO  F i g u r e 15. ELISA r e a c t i v i t y o f AlO TsF p u r i f i e d o v e r P815-48 columns. AlO a s c i t e s was passed o v e r and e l u t e d from a column t o which P815 membrane e x t r a c t s had been l i n k e d .  E l u a t e s were c o a t e d i n ELISA p l a t e s t o  assay f o r r e a c t i v i t y t o t h e B16G monoclonal. o f AlO a s c i t e s e l u t e d from P815-4B; ( 0 over P815-4B; (  ( x  x ) , ELISA  reactivity  0 ) , BW5147 a s c i t e s " p u r i f i e d "  ) AlO a s c i t e s " p u r i f i e d " o v e r a column t o which  normal DBA/2 s p l e e n e x t r a c t s were l i n k e d .  65  V  AVEUGE IUMOI  »  U T S POST Pfl 13 INJECTION »  Figure 16A.  Growth of P815 tumors In DBA/2 mice injected i.v. with 25.0  pg of AlO or G10 (NH) SO -precipitated ascites fluid or PBS. There were between 8-10 animals in each group.  A "b" superscript over a  data point indicates significance to 99% (Student's t-test). is given in mm  Tumor area  Figure 16B. Survival curves of mice treated with 20 yg AlO i.v. 3  concurrent with a dose of 5 x 10 P815 cells s.c. ( x-• of AlO treated mice; ( 0  x ), survival  0 ), survival of PBS control mice; ( 0  survival of mice irradiated with 100 rads prior to tumor dose (immune impaired).  Eight DBA/2 female mice (4-6 weeks old) were used in each  group of the experiment.  0  67  Figure 17. DBA/2 mice.  In vivo activity of affinity purified AlO TsF in P815-bearing Animals (8 per group) received 20 pg AlO TsF concurrent 4  with a s.c. flank injection of 10  P815 tumor cells.  ( x  x ), animals  receiving 20 pg AlO TsF purified over B16G-4B columns; ( •  • ), animals receiving AlO TsF purified over P815-4B columns;  ( 0  0 ), PBS control animals.  Differences between suppressed and  control mice were significant >.98 on a l l days after day 7.  68  Further experiments were carried out to assess the dose response of animals to Bl6G-reactive AlO material. Mice were injected i.v. with varying concentrations of B16G eluted AlO TsF along with appropriate controls on the same day that they received 1 x 10  P815 cells s.c.  The  results (Fig. 18) show that those mice receiving the highest dose of the AlO TsF (20 yg per mouse) demonstrated the greatest degree of enhanced tumor growth, but there was also a significant enhancement in mice receiving concentrations of affinity purified AlO as low as 200 ng.  These  51 results were repeated in an in vitro CTL k i l l i n g assay (Cr  release)  (Figure 19). Specificity.  We have shown that immunoadsorbed AlO ascites fluid not only  binds to P815 immunoadsorbents but also enhances the growth of this tumor in DBA/2 mice, thus f u l f i l l i n g criteria for a factor which binds tumor antigen as well as suppressing immune responsiveness to the tumor in vivo.  However, we had not shown that the AlO TsF was specific for the  P815 tumor in i t s in vivo action. Accordingly, experiments were carried out to assess the possible immunosuppressive properties of AlO ascites. Initially, the ability of B16G-absorbed AlO ascites to effect the MLR of DBA/2 splenocytes with BIO.BR irradiated splenocytes was tested, since this assay was used i n i t i a l l y to demonstrate that B16G absorbed out a variety of suppressive molecules from DBA/2 splenocytes.  The results  (Fig. 20) show clearly that Bl6G-reactive material from AlO does not inhibit this reaction.  69  •  J  S  H DAYS POST  3  ij  17  INJKTION*  Figure 18. Effect of different dose levels of AlO Bl6G-affinity purified material on the growth of P815 tumors in DBA/2 mice. from 20 yg (undiluted) to 20 ng (1:1000).  Dose levels ranged 2  Tumor area i s given in mm .  A "b" superscript over a data point indicates statistical >.98.  significance  Figure 19.  In vitro activity of AlO TsF in a chromium 51 release assay  (CTL killing).  AlO TsF purified over B16G-4B was added at various  concentrations at 3 different effector-target ratios to cultures containing P815 primed DBA/2 splenocytes and Cr"'* labelled P815 tumor 51 target cells.  ( Q  & ), % suppression of Cr  release caused by  AlO TsF added to cultures containing splenocytes and tumor cells at a 10:1 ratio; ( x — x ), AlO added to 5:1 E:T ratio cultures; ( •  • ), AlO  TsF added to 1:1, effector target ratio cultures; ( 0---0 ), effect of undiluted BW5147 B16G eluate added to a E:T 10:1 culture; ( 0——0 ), effect of BW5147 eluates added to E:T = 5 cultures. exceeded 10% of the mean in this assay. of octuplicate wells.  Standard error never  Mean values represent the average  Mmuu. MLC  >/• DILUTION  i/ia or  n«a  t/S2  ima  C L O A T M  Figure 20. The effect of affinity purified AlO material on the MLR between DBA/2 splenocytes and BIO.BR targets.  Control cultures were  cultured with equivalent fractions eluted from B16G column after G10 hi been absorbed. No AlO dilution ( • ), or G10 dilution ( O ), gave a result significantly different from the normal MLC ( B ). This result demonstrates the specificity of AlO TsF; i t will only inhibit P815-specific immune responses. This type of experiment was performed twice.  72  Subsequently, experiments were carried out to determine whether the AlO TsF could influence the growth of other syngeneic tumor cells in DBA/2 mice; namely, the L1210 leukaemia, and the M-l rhabdomyosarcoma. Experiments testing the effect of Bl6G-eluted AlO material on the growth of these tumors were carried out in a manner analogous to the above studies.  The results (Fig. 21 and Table II) show clearly that the AlO TsF  preparation used, while preventing the immune response to P815, has no effect on the response to either L1210 or M-l.  Biochemical characterization.  The native molecular weight of the AlO TsF  was determined by passage of AlO ascites over a Sephadex G-150 column. The elution profile was monitored for material absorbing at 280nm, as well as for reactivity with B16G (Fig. 22).  The results show that while a  majority of the 280nm absorbing material is in the molecular weight range of 70 kD (presumably albumin) the major region reacting with B16G in the ELISA is of a higher molecular weight range (in the region of 80 kD) (Fig. 22).  When the ELISA-reactive material was tested in vivo for Its  ability to enhance P815 growth in DBA/2 mice, along with appropriate controls, i t was found that this material was capable of enhancing tumor growth (Fig. 23). Further characterization of the AlO TsF was carried out on AlO material eluted from B16G columns with PAGE. The reduced M.W.  of the TsF  was determined by SDS-PAGE. Under reducing conditions (.1% DTT), the molecule was observed to run mainly as a heterodimer of subunit 45 kD, and 25 kD, respectively (Fig. 24).  Material eluted from the 45 and 25,000 MW  Page 73 m i s s e d  in  numbering  Table II.  Average tumor size of mice receiving AlO TsF and various tumors  Experimental Group Day after  +10  Tumor  P815 cells  4  3 x 1Q L1210 cells  + 1Q M-l cells  3  AlO  PBS  + AlO TsF  5  + PBS  + AlO TsF  + PBS  Injection 8 9  4.3  +  2.5 b  6.1  5.0  +  2.1  6.0  +  1.6  8.0  +  2.3  8.2  +  2.1  +  2.5  10  +  4.0  49  +  14.1  44  +  10.7  4.5  10  23  +  11  68  +  16.7  33  +  13.2  72  +  20.7  77  +  18.7  13  12  111  +  27.3  43  +  17.1  108  +  31.0  116  +  28.1  24  13  130  +  32.0  60  +  23.8  137  +  39.3  135  +  32.7  160  +  45.8  161  +  38.9  b  b  b  14  +  2.6  5.0  +  2.6  4.6  16.2  +  4.8  +  8.4  25  +  7.4  28  +  9.8  37  +  10.9  95  +  33.0  98  +  28.8  5.0  Average tumor size (mm ) ± standard deviation of DBA/2 mice injected with either P815, L1210, or M~l.  Each  group contained eight mice that had received either AlO (20 pg) or PBS i.v. on the day they received tumor cells subcutaneously. "P =  0.05 by Student's t-test.  75  F i g u r e 22.  E l u t i o n p r o f i l e o f AlO a s c i t e s o v e r Sephadex G-150; shown a r e  280 nm a b s o r p t i o n ( 0—-O f r a c t i o n 27, 150,000 MW,  ) as w e l l as B16G ELISA r e a c t i v i t y ( 0 — 0 f r a c t i o n 40, 70,000 MW,  f r a c t i o n 50, 40,000  Column volume was 74 m i s ; 3 mis o f a 5 mg/ml AlO a s c i t e s s o l u t i o n was applied.  ) MW.  76  DAYS  Figure 23. TsF  POST ' 8 1 3  INJECTION*  The effect of Bl6G-reactlve fractions from G150-purifled AlO  on the growth of P815 tumors in DBA/2 mice.  material from fraction 37 (Fig. 22) ( 0  Mice received 20 pg of  0 ), or 20 pg material from  fraction 37 of a G10 ascites G150 run (no ELISA reactivity) ( 0  0 ).  1  \  3  Figure 24. SDS-PAGE analysis of AlO TsF. AlO ascites was purified over B16G columns, dialysed against d i s t i l l e d HgO, lyophilized and run under reducing conditions on a 10% SDS-PAGE gel. Gel was silver stained.  Lane  1, ovalbumin, MW 43,000; Lane 2, AlO ascites "purified" over an irrelevant monoclonal column; Lane 3, AlO ascites purified over B16G-4B. For purposes of SDS-PAGE analysis, only peak KLISA-reactive fractions from B16G columns were run (e.g. Fraction 4, Fig. 15).  78  bands tested positively for reactivity to B16G in an ELISA assay (Fig. 25). For this assay, proteins were eluted from the gel as in Chapter I. More recently, in vivo competition experiments between B16G and AlO 4  TsF were performed. Mice were injected with 10 P815 tumor cells s.c. as before.  One group received 20 yg AlO TsF as previously described.  However, the other experimental group received a concurrent i.v. injection of 20 yg AlO TsF and 100 yg B16G MAb. As can be seen in Figure 26, this group, while experiencing more rapid tumor growth than control mice, demonstrated significantly slower tumor growth relative to AlO treated mice.  These results indicate that addition of B16G (suppressor deletion)  and AlO TsF (suppressor augmentation) are directly competing effects, especially i f one notes what effect the addition of 100 yg B16G usually has (20% survival, significantly reduced growth relative to control mice) (112). DISCUSSION  The probe used in this study was the MAb, B16G. Since studies with B16G indicated that this "anti-TsF" MAb was directed to a constant region of a DBA/2 immunuregulatory protein, and since i t was capable of abrogating immunosuppressive effects in a variety of assays (112,120,121), B16G was used in this study as a probe to identify T cell hybridomas secreting detectable levels of Bl6G-reactive materials.  Fusions were  carried out using the c e l l line BW5147 and splenocytes from DBA/2 mice which had been sensitized 4 days earlier with membrane extracts from P815  79  IO  I0O RECIPROCAL  OF  SUBUNIT  O O O DILUTION  IOOOO »  Figure 25. ELISA reactivity of AlO TsF subunits eluted from SDS-PAGE. AlO TsF purified over B16G-4B columns was dialysed (against d i s t i l l e d HgO) lyophilized, and run in 10% reducing SDS-PAGE. Bands were visualized with silver stain, cut out, and proteins eluted with carbonate buffer.  The 45 kD band ( •  -9 ), and 25 kD band ( x  -x ) were eluted  and coated on ELISA plates for analysis of reactivity to B16G. Bands from the "45 kD" region of a lane In which BW5147 ascites "purified" over B16G-4B had been run were similarly excised and tested in the ELISA ( O shown).  • ).  No other region in the gel gave ELISA reactivity (not  80  Figure 26.  Inhibition of AlO TsF suppressive activity by in vivo  administration of B16G monoclonal. DBA mice receiving AlO TsF; ( x  ( x  x ), growth of P815 tumor in  x ), growth of tumor in DBA mice  receiving AlO TsF and B16G Ab concurrently; ( 0 control mice.  0 ), growth of P815 in  81  cells.  This protocol has been described earlier as one which stimulates  the production of tumor specific thymic  T3C  in t h i 3 system (111,112). A  single hybridoma out of over 3,500, AlO, was identified as one which secreted relatively large quantities of B16G-reactive material as detected by ELISA. The relatively insensitive procedure used in selection, while being labor intensive, selected only clones secreting comparatively amounts of B16G-reactive material.  large  A series of immunoadsorbent studies  established that AlO (either in culture or as an ascites tumor) secreted material which could be specifically purified over Bl6G-Sepharose columns and not over irrelevant columns, and that Bl6G-negative T c e l l hybridomas tested under identical conditions, did not.  Further immunoadsorbent  studies showed that B16G-reactive material could be eluted from P815 membrane extract-Sepharose columns, indicating that AlO might be secreting a TsF capable of binding to P815 antigens, and thus with specificity for the P815 tumor system. In vivo experiments in which affinity purified AlO putative TsF were injected along with appropriate controls showed that mice treated with AlO TsF demonstrated significantly accelerated growth of P815 over control animals, and that the effect appeared to be  do3e  dependent.  It is worth  noting that concentrations of AlO as low as 200 ng per mouse had a significant effect on tumor growth.  The specificity of this effect was  demonstrated by the observation that AlO-injected mice did not exhibit accelerated growth of the L1210 or M-l tumors in comparison to appropriately injected control animals.  82  The assumption that affinity purified AlO material i s not non-specifically immunosuppressive was further established by demonstrating that i t did not inhibit the MLR between DBA/2 splenocytes and BIO.BR targets.  The generation of CTL's to P815 tumor cells was,  however, suppressed.  The possibility that AlO material might have a  direct effect in vitro on the growth of P815 cells was also investigated, and i t was found that this was not the case and could therefore not account for the accelerated growth of P815 in DBA/2 mice (data not shown). Biochemical characterization of the AlO active component has shown that i t has a native molecular weight i n the region of 70 - 80 kD, but when affinity purified material is run on reducing SDS-PAGE i t i s visualized as a heterodimer with molecular weights in the region of 45 kD and 25 kD. Frequently, as in the case of HTsF, 80 kD bands, representing the native MW of the TsF molecule are observed on the gels (Chapter III). Further studies on the AlO molecule w i l l involve purification and serological characterization of individual subunits.  Since B16G reacts  with both the 45 and 25 kD subunit (though with 1000-fold greater affinity for the former), i t i s possible that the 25 kD molecule represents a de-glycosylated form of the 45 kD subunit, or that each represents one unit of the complete (70 kD) TsF protein and shares constant region homology (like the H and L chains of the Ab molecule).  Since the AlO  hybridoma secretes considerable amounts of this molecule, further research in this lab will be directed towards sequence analysis of the subunits. Both amino acid analysis and tryptic peptide mapping of the AlO TsF molecule have already been performed.  Table III. Comparison of AlO TsF with various antigen-specific TsFs  Antigen specificity  Source  MHC Restriction  Presence of CHO?  MW  Structure  Investigator and reference ( )  GAT  Hybrid 258C4.4  none  29 kD  1 chain  Kapp (39)  GT  Hybrid 395A4.4  none  70 kD  2 chains  Kapp (39)  SRBC ClLy23/4  T-cell line  ND  70 kD  2 subunits 45 kD and 25 kD  Cantor  KLH  Hybridoma  I-Jb  ND  68 kD  2 chains  Taniguchi (114)  NP  Hybridoma  I-J  ND  ND  ND  Bennacerraf  P815 Ag (AlO)  Hybridoma  ND  70 kD  2 subunits 45 kD and 25 kD  Levy (141)  (113)  (135)  84  Chapter Summary  In previous publications and Chapter I, we have described a monoclonal antibody, B16G, which has been shown to bind to suppressive T c e l l factors in DBA/2 mice.  Therefore, B16G was used as a probe to  identify T c e l l hybridomas secreting putative TsFs.  Hybridomas were  obtained by the fusion of DBA/2 thymocytes stimulated in vivo by P815" tumor membrane extracts with the thymoma BW5147. One such hybridoma, AlO, was selected and used for further studies. From both the supernatants, and ascites fluid of this hybrid could be obtained a factor which could specifically bind to both B16G and P815 antigen immunoadsorbent columns, and which scored positively with B16G in the ELISA after elution.  Such  reactivity could not be obtained from AlO supernatants or ascites absorbed over irrelevant columns, nor was i t obtained from supernatants or ascites from other T c e l l hybrids which had scored Bl6G-non-reactive in the original screening. In vivo studies indicated that affinity purified AlO material injected into DBA/2J mice enhanced significantly the growth of P815 tumor cells, but not the growth of other DBA/2 syngeneic tumor lines such as L1210 or M-l.  Additionally, this material did not inhibit the in vitro  MLR between DBA/2 splenocytes and allogeneic BIO.BR target cells (unlike B16G purified material from whole DBA/2 spleens, which was shown to be suppressive in this type of MLR). Biochemical analysis of t h i 3 tumor-specific TsF from AlO was undertaken; the native molecular weight was found to be in the region of  85  70 - 80 kD.  Under reducing conditions, affinity-purified AlO TsF was  found to resolve partially in SDS-PAGE as an apparent heterodimer of 45 kD and 25 kD, respectively.  It has not been proven unequivocally that the 45  and 25 kD peptides seen under reducing conditions are subunits linked by disulfide bonds, or whether they constitute degraded breakdown products of the 80 kD structure.  86  Chapter III  Characterization of an anti-idiotypic TsC,, hybrid analogue involved in the regulation of the immune response to the P815 mastocytoma  INTRODUCTION  There are a number of models which explain the down regulation of the immune response. While there are some differences between the different models based on the experimental systems in question, certain basic assumptions are common to a l l .  It is generally agreed that  immunosuppression involves a cascade of cellular interactions in which three distinct T c e l l subsets are involved and designated here as Tsl, Ts2 and Ts3 (herein we use the nomenclature used by Greene and co-workers (126).  There are a number of experimental models in which f i r s t and  second order TsC have been clearly demonstrated (127-130), as well as those in which Tsl has been shown to be antigen specific ( i d ) and Ts2 +  to be anti-idiotypic (126,131,132).  Phenotypically i t i s also generally  agreed that Tsl are Lyl and I-J , Ts2 are anti-idiotypic, Lyl,2 and I - J and Ts3 (the effector) are i d , Ly2 and I - J (126). In +  +  +  +  most systems which have been investigated in depth i t has been shown that both Tsl and Ts2 secrete factors which can replace the requirement for the cell in i t s regulatory capacity.  These networks have perhaps been most  extensively studied in the anti-azobenzene arsonate (ABA) delayed type hypersensitivity suppressor cascade. Mice immunized with ABA (125) hapten  87  conjugated chemically to syngeneic thymocytes results in the generation of a Tsl cell population ( i d which will block the DTH response to ABA +  (6).  These cells, like the Tsl population discovered in the P815 system,  were Lyt l 23 +  (126).  The cells and the factors they produced were  antigen specific in their suppressive action, bound antigen, and bore idiotype (134), a l l characteristics shared with AlO TsF.  TsFl could be  used to induce a Ts2 population (135) which did not bear idiotype, but would bind to idiotype bearing columns or coated plates (therefore anti-idiotypic in nature) (136).  From these Ts2 cells, a TsF2 soluble  factor could be isolated, with the same binding properties; like Tsl cells and factors, these were suppressive in an antigen-specific manner (137). More recently, this work was repeated with factors derived from hybridomas created by the fusion of these cell hybridomas to BW5147 (138).  With  these observations in mind, the experiments detailed in this Chapter were performed.  AlO TsFl was used to stimulate syngeneic mice; subsequently,  their thymocytes were fused to BW5147. Hybridomas  thu3  produced were  screened for TsF2 production on idiotype (AlO) coated ELISA plates. One such hybrid, A29, was raised by this method.  Its functional  characteristics are described below; like AlO, A29 TsF can cause tumor-specific Immune suppression, has similar biochemical characteristics, and similar serology; unlike AlO TsF, i t s binding target is the idiotype of TsF^, not tumor antigen. We propose, therefore that the A29 cell line represents the hybrid analogue of the Ts2 cell population in the suppressor network regulating the response to P815.  88  MATERIALS AND METHODS  Experimental Animals. Female DBA/2 mice and CBA/Balb/c Fl mice 6-8 weeks of age were used in a l l experiments.  Cell Lines.  The P815 mastocytoma of DBA/2 mice the AlO hybridoma and the  BW5147 line were maintained in this laboratory as described in Chapter II. Another T cell hybridoma, Fdll, which secretes a TsF binding the ferredoxin molecule was also used; this hybrid was raised in our laboratory.  Linkage of AlO TsF to Sepharose-4B.  The AlO TsF was produced by growing  the AlO hybridoma as an ascites tumor in CBAxDBA/2 Fl mice (Chapter II). AlO TsF was linked to Sepharose 4B beads by cyanogen bromide linkage according to standard procedures. linked.  1.0 mg AlO TsF per ml beads was  The columns thus made retained the AlO idiotype; this was  verified by the observation that AlO TsF bound to Sepharose could s t i l l adsorb an anti-id AlO monoclonal antibody, D110C, produced in this laboratory.  Kinetic Studies on TsF Production in Tumor Bearing Mice. In order to determine whether increased levels of TsC could be detected in tumor bearing mice, and whether evidence could be obtained for the development of a putative anti-idiotypic Ts2 in this system, the following experiments were carried out. Three selective immunoadsorbent columns were prepared  89  by linkage of materials to cyanogen bromide activated Sepharose 4B. The materials Included; the MAb B16G, P815 membrane extracts (both conjugated at 5.0 mg/ml beads), and the AlO molecule purified as described above and conjugated at 1.0 mg/ml beads.  Control columns containing irrelevant MAb,  and control ascites passed over B16G columns were also prepared. 4  A group  of 25 mice were injected subcutaneously with 10 P815 cells in the right flank.  On days 1, 2, 4, 6, 7, 9 and 12, three mice were sacrificed and  their spleens removed and pooled.  Untreated control mice were sacrificed  and their spleens treated in the same manner throughout.  Spleen cells  were washed with PBS and solubilized using a standard lysis buffer (containing .5% NP40).  Soluble materials from both tumor bearing and  control mice were passed i n i t i a l l y over a B16G immunoadsorbent, and reactive material was eluted with 0.1N HC1. Aliquots of each fraction (containing polyclonal TsF) were titrated at doubling dilutions in bicarbonate coating buffer onto ELISA plates.  The remaining  TsF-containing eluates were then passed over P815 antigen columns, and reactive material (presumed to contain P815-specific Tsl) was eluted. Again aliquots from each fraction from tumor bearing and normal mice were titrated on ELISA plates as described.  Finally, remaining eluted  materials were passed over the AlO immunoadsorbent column.  Reactive  material (containing putative anti-idiotypic components) wa3 eluted and tested In the ELISA.  These procedures were repeated on a l l the days given  above for both tumor bearing and control animals (see Figure 27). o  ELISA  plates were left for 18 h at 4 C after coating and were developed using purified B16G MAb at a concentration of 10 yg/ml in PBS-tween.  Final  90  development of plates was carried out using alkallne-phosphatase labeled rabbit anti-mouse Ig.  Reactivity over background levels was determined by  comparing the values obtained for materials from tumor bearing animals to the equivalent control value.  Production of the A29 T Cell Hybridoma.  The procedure described in  Chapter II for cell fusion of DBA/2 splenocytes with the BW5147 c e l l line was used. DBA/2 mice were injected l.p. on day 0 with 20 yg of affinity purified AlO (to induce Ts2 production) and on day 6, animals were sacrificed, their thymus and spleen being used for fusion with BW5147 cells.  Fusions were carried out as described previously using  polyethylene glycol and selection In HAT medium. The screening for a T cell hybridoma producing a factor anti-ldlotypic for AlO was done as follows. Hybridomas were i n i t i a l l y plated onto microtitre wells and when obvious growth of clones was visible, supernatant aliquots were tested in a double sandwich KLISA. ELISA Immulon II plates were coated with affinity purified AlO at a concentration of 20 yg/ml.  Culture supernatants were then plated onto  these ELISAs, allowed to bind for 2 h, washed and developed with B16G. Initially, l t was thought that this screening procedure would result in a negative selection; i.e., the anti-idiotypic molecule would sterically interfere with subsequent binding of B16G to AlO and thus ELISA reactivity would be lower In those wells containing reactive material. In fact, the reverse was observed in that A29 anti-idiotypic TsF enhanced the ELISA  91  reactivity.  The reasons for this became apparent later since i t appears  that the anti-idiotypic material is a l 3 0 reactive with B16G.  A total of  2,500 hybrids were screened in this way but only three proved to be a consistent high producer of material which bound to the AlO molecule.  One  of these, the hybridoma A29, was cloned three times, and has been maintained in vitro in DME plus 20% FCS with a continuous feeder layer of DBA/2 splenocytes since i t was isolated.  This hybrid is not capable of  independent growth and appears to have a requirement for either a dense feeder layer or conditioned medium containing PMA-stimulated EL4 supernatant.  Purification of A29 (TsF2).  The soluble product from the A29 hybridoma  was purified from culture supernatants or from ascites by affinity purification on either B16G-Sepharose or AlO-Sepharose columns as described.  This hybridoma grows slowly and produces only limiting amounts  of reactive materials in culture (2 yg TsF/1), but workable quantities of A29 TsF (10-20 yg/ml) may be obtained from ascites.  A29 In vitro Experiments.  Bl6G-affinity purified A29 TsF from ascites was  injected i.v. into groups of DBA/2 mice at concentrations of 20 yg/mouse. 4 At the same time they were inoculated s.c. with 10 P815 cells in the right flank.  Control groups received PBS at equivalent volumes. Mice  were monitored daily to assess tumor growth.  Tumor size was determined by  squaring two cross sectional measurements using calipers and death times were recorded.  92  Panning.  Plastic petri plates (Falcon 3003) were coated overnight with 5  ml of PBS containing 16 yg/ml of affinity purified A29 or control material from BW5147 cells treated in an analogous manner.  The plates  were washed 3 times with PBS, blocked for 1 hr at 4°C with 10% FCS in PBS and again washed with PBS. DBA/2 spleen cells were washed, resuspended in DME plus 5% FCS, added to the plates at 5x10 cells per plate, and incubated for 1 h at 4°C The nonadherent cells were removed, counted and assayed in the CTL microassay as described below.  Intracellular Calcium Flux.  Changes of [Ca^ ] within AlO or A29 +  hybridoma cells when their receptors were bound by either B16G or the appropriate TsF were measured by the method of Tsien et a l . (139). Q  Briefly, AlO (or A29) cells at a concentration of 10 cells/ml were incubated with 50 um Quin 2 acetoxymethyl ester (Quin 2/AM) at 37°C, 10% C0 , in DME for 20 min. This mixture was then diluted 10-fold and 2  incubated for a further h. 1 ml aliquots of this stock solution 7 (containing  10 cells) were then collected, the cells spun down, and  washed once with PBS. For measurement of fluorescence, the cells were resuspended in 1.0 ml modified PBS (containing 1 mM GaCl,, added to standard PBS) and transferred to a quartz cuvette. in a Perkin-Elmer  Analysis was performed  650 - 10S fluorescence spectrophotomer.  emission wavelengths used were 339 and 492 nm respectively.  Excitation and Fluorescence  was calibrated by release of Quin 2/AM from the cells by addition of .02% (v/v) Triton X-100.  Background fluorescence of the monoclonals used -  B16G and 3.65 H (irrelevant) - was assayed by adding stimulatory  93  concentrations of the antibody to cuvettes containing only PBS + 1 mM CaCl^j or to cuvettes containing AlO cells not loaded with Quin 2/AM. Concentrations of B16G antibody and TsF that were found to be maximally stimulatory (and are reported here) were 30 yg/ml and 5 yg/ml, respectively. level.  Control Ab and TsF's were administered at the same dose  94  RESULTS  Our Initial studies with the AlO T c e l l hybridoma and i t s soluble antigen binding factor lead us to believe that this cell line represented a prototype for a Tsl in the suppressor cell circuitry controlling the response to P815.  Further experiments were carried out to determine  whether we could provide evidence for other cells in this cascade and whether these would be reactive to AlO (i.e., anti-idiotypic).  To this  4  end, DBA/2 mice were injected s.c. with 10 P815 cells, and groups of three animals were sacrificed sequentially, their spleens removed, the cells lysed, and the soluble extract passed sequentially over three immunoadsorbent columns as diagrammed in the flow chart (Fig. 27). Material eluting from the f i r s t column (B16G) which should catch a l l putative TsF was subsequently passed over a P815 column (to catch specific TsF^) and then over an AlO column (to catch anti-idiotypic TsF^). Materials eluting from a l l columns were quantified by recording their absorbence at 280 nm and titrated under equivalent conditions for reactivity with B16G in an ELISA system.  Control material from untreated  mice was run at the same time and provided the base level in a l l materials.  The results (Fig. 28) are presented as ratios of ELISA  reactivity obtained from materials derived from either treated or untreated animals.  It can be seen that total TsF content within the two  groups does not change significantly throughout the course of tumor growth.  However, TsF which specifically binds P815 membrane extracts  increases in tumor bearing animals until about day 4 after which elevated  95  FLOW CHART FOR CELL FLUX EXPERIMENTS  16 DBA MICE RECIEVE NO TUMOR CELLS (CONTROL)  „  RECIEVE AN INJECTION OF 10 P815 CELLS a . c .  SACRIFICE 3 MICE FROM EACH GROUP ON DATS 1,2,4,6,7,9,and 12  REMOVE SPLEENS AND SOLUBILIZE MEMBRANE PROTEINS BY DETERGENT LYSIS  PURIFY WHOLE POLYCLONAL TsF BY LWMUKOSORPTION ON B16C COLUMNS  COAT ELUATES ON ELISA PLATES: ANALYSE FOR B16G REACTIVITY  PURIFY ANTI-P815 TaP BY IMMUNOSORPTION ON P815 ANTIGEN COLUMNS COAT ELUATES ON ELISA PLATES; ANALYSE FOR B16G REACTIVITY PURIFY ANTI-ID (TaF2) BY IMMUNOSORPTION ON AlO TaF COLUMNS COAT ELISA PLATES WITH COLUMN ELUATEj ANALYSE FOR B16C REACTIVITY  Figure 27. Flow chart describing the method used to analyse splenocytes of DBA/2 mice bearing P815 tumors for their relative level of total TsF, TsF  (id) and TsF  (anti i d ) . Results are shown in Figure 28.  96  Figure 28.  TsC c e l l levels In tumor bearing DBA/2 mice.  Spleens of P815  tumor-bearing mice were removed on days 1, 2, 4, 6, 9 and 12. sacrificed on each day.  ( O---0  ), polyclonal TsF isolated from tumor  bearing spleens passed over B16G-4B; ( • isolated from P815 Ag-4B; ( a  A ), TsF  Isolated over AlO TsF-4B columns. of ELISA values at  0  D  ^  0  t j  3 mice were  • ), TsF^ (anti-P815 TsF) 2  (aid aA10 TsF)  A l l values are expressed as the ratio  compared to control values from non-tumor  bearLng mice whose spleens had been treated equivalently.  97  levels remain constant through the course of the experiment.  The most  surprising result found here was that the level of anti-idiotypic T3F2 (AlO binding) material increases dramatically in the early course of tumor growth until about day 7 after which levels drop off and regress rapidly toward normal levels.  These results indicated the validity of further  experiments in an attempt to isolate anti-AlO (anti-idiotypic) T suppressor c e l l hybridomas. Because i t appeared that an anti-idiotypic TsF2 might be stimulated by the proliferation of Tsl cells and release of i d TsF^, DBA/2 mice +  were injected i.p. with affinity purified AlO (20 yg) 5 days prior to sacrifice and fusion of their thymocytes and splenocytes with BW5147 cells.  Hybridoma supernatants were tested in a sandwich ELISA for the  presence of AlO-binding material reactive with B16G.  Over 2,500  hybridomas were screened and one, A29 was found to secrete material which in the screening procedure used, enhanced B16G binding (Fig. 29).  Further  tests on A29 TsF in the sandwich ELISA revealed the following results: A29 TsF binds specifically to AlO TsF coated plates enhancing Bl6G-reactivity above background (Fig. 29, columns 3, 9, 10) but will not bind to plates coated with Fdll TsF (an irrelevant TsF directed to Fd Ag) (column 4).  AlO TsF, or other irrelevant TsF's applied to AlO coated  plates will not bind and enhance the ELISA (columns 2, 7, 8, and 11). These results, as did the c e l l flux studies, indicated that this anti-idiotypic factor shared common features with the AlO molecule, since they both reacted with the B16G MAb. Subsequently, A29 was cloned, expanded, and maintained either in tissue culture, or grown as ascites in  98  Figure 29.  A29 sandwich ELISA assay.  Columns 1 - 5  A29 TsF from culture  supernatant was applied to plates coated with AlO TsF (column 3) or Fdll TsF (irrelevant TsF - column 4).  Controls include:  no TsF added (gives  background) column 1; AlO TsF added to AlO coated plates, column 2; AlO T3F added to Fdll TsF coated plates, column 5.  Columns 6 - 1 1 : A29 TsF  from ascites applied to AlO TsF coated plates (column 9, 1/100 dilution, column 10, 1/300 dilution); Columns 6, no TsF added, (= background), columns 7, 8, and 11, addition of AlO T3F, DC-10 TsF or Fdll TsF ascites, respectively to the AlO coated plates, a l l at 1/100 dilution.  Extra TsF  bound to the AlO coated plates was a33ayed for by the addition of B16G MAb and RaMIg-AP as described in Methods.  99  C O L U M N FRACTION- -  Figure 30.  •  Purification of A29 (aid TsFg) over AlO T3F 4B columns.  A29 ascites was passed over and eluted from a column to which AlO TsF had been bound.  Eluted adherent material was coated on ELISA plates for  analysis of reactivity to B16G monoclonal ( x — — x ), ELISA reactivity of A29 ascites purified over AlO TsF-4B; ( 0 ascites eluted from the AlO column.  0 ), reactivity of BW5147  100  pristane treated, irradiated (CBA x Balb/c) Fl animals.  Material from A29  ascites was affinity purified over either AlO or B16G affinity columns, and examined for reactivity in ELISA with B16G.  One such set of data is  shown in Figure 30, in which A29 was affinity purified over an AlO column and tested in ELISA with B16G. from the parent BW5147 line.  The control material used here was ascites Only material eluting after A29 was passed  over the AlO column contained significant B16G reactive material thus establishing the anti-idiotypic nature of A29 and demonstrating that AlO was not leaching non-specifically from the column.  Figure 31 shows the  gel pattern of A29 (lane C) purified over B16G and demonstrates that A29 TsF exhibits a pattern indistinguishable from AlO TsF (lane A), that of a major band at about 70 kD.  Low molecular weight bands at <10,000 daltons  represent either break-down products or non-specific materials eluting from the column, and are frequently seen with AlO TsF preparations as well. In order to determine whether reactivity to both AlO and B16G  could  be detected by surface binding of these molecules to A29, studies on 2+ intracellular Ca  flux were set up.  The results (Fig. 32) show  unequivocally that both affinity purified AlO and B16G cause significant changes in calcium concentrations at the membrane level of A29 whereas appropriate control materials do not.  Feeder layer cells (which may  be  present in the A29 preparations) do not show reactivity when tested alone (not shown).  Similarly (Fig. 33), affinity purified A29 factor and 2+  both cause significant Ca material does not.  B16G  flux within AlO cells whereas control  These results implicate equivalent stimulation between  101  A  Figure 31.  B  D  SDS-PAGE a n a l y s i s o f a f f i n i t y p u r i f i e d A29 T s F .  p u r i f i e d over B16G-4B columns from a s c i t e s , PAGE g e l .  C  l y o p h i l i z e d and r u n on a 10%  G e l was d e v e l o p e d w i t h Coomassie B l u e .  p u r i f i e d over B16G;  M a t e r i a l was  Lane A ,  Lane B, BW5147 " p u r i f i e d " over B16G;  Lane D, low MW s t a n d a r d s r a n g i n g from 30 - 93 kD.  AlO  TsF  Lane C, A29  In t h i s r u n , both  and A29 TsF appear i n t h e i r n a t i v e c o n f o r m a t i o n o f 70  kD.  TsF; AlO  p 102  z Ui  11  8  13  15  17  T I M E [mini  Figure 32.  Intracellular calcium flux within A29 hybrid cells stimulated  with AlO TsF or B16G MAb.  1.0 min, stimulation of A29 cells with 5  yg/ml irrelevant TsF (A29 TsF or aFd TsF); 5.0 min, stimulation of A29 with AlO TsF (equivalent concentration to controls); 9 min, stimulation 30 ug/ml with 3.65 H Irrelevant MAb; and 13 min, stimulation of A29 with 30 pg/ml B16G monoclonal.  103  UJ  U Z  LU  M LU  CC  o  5  F i g u r e 33. A29 with  8  TIME (min)  I n t r a c e l l u l a r calcium  T 3 F or B16G  monoclonal.  i r r e l e v a n t TsF at 1.0  or with A29  TsF  II  f l u x w i t h i n AlO  C e l l s loaded min  (at 5 m i n u t e s ) .  14  with  (AlO TsF or ant  (14 minutes).  f l u o r e s c e n c e at 492  Ca^ nm.  +  c e l l s stimulated  Quin-2 dye were  C e l l s were a l s o c h a l l e n g e d  I n f l u x (and b i n d i n g  with  measured by  In which TsF or Ab  reproducible.  Fdll)  H (10 minutes) or  t o Quln-2) was  Regardless o f the o r d e r  added to the c e l l c u l t u r e s , r e s u l t s were  3.65  with  stimulated  a n t i - f e r r e d o x i n TsF,  e q u i v a l e n t doses o f i r r e l e v a n t mnoclonal a n t i b o d y B16G  17  was  104  id  +  and id  cell populations which supports a concept of connectivity  between these two cells. In an attempt to determine whether any biological activity on the part of the A29 affinity purified material could be demonstrated, i t s effect on the in vitro generation of CTL specific for P815 determined.  was  Results of one such experiment (Fig. 34) show that addition  of A29 material to in vitro cultures did cause a suppression of CTL activity, whereas equivalent control material did not.  This effect is  demonstrably specific since A29 material has been shown to lack a generally immunosuppressive effect (data not shown).  Further, A29  affinity purified material was coated onto plastic petri dishes and used for panning DBA/2 splenocytes prior to culturing them for the generation of CTL specific for P815.  The results (Fig. 35) show that such a  procedure gives rise to cells capable of generating a significantly higher CTL response to P815 cells than appropriately treated controls. These results indicate that cells for which A29 is anti-idiotypic (i.e., i d  +  cells) are capable of down regulating this response. Attempts to demonstrate the in vivo effects of A29 met with mixed results.  In a majority of the experiments done, when A29 was injected  i.v. either on the day of tumor c e l l administration or on day  +2,  accelerated tumor growth (Fig. 36) and death (Fig. 37) was seen. However, i t should be mentioned that in 2 of the 6 experiments of this nature done, a reverse effect was seen in that enhanced tumor resistance (in some individuals within the group) over appropriately treated controls was observed (data not shown). The implications of these effects are discussed below.  contrasuppressive  105  CONCENTRATION OF FACTOR(ug/ml)  51 Figure 34. killing).  A29 TsF is suppressive in a Cr release assay (CTL Affinity purified A29 ( • ) or BW5147 ( • ) was added at  1.0, and 0.5 yg/ml to CTL from DBA/2 spleen cells against P815 cells, using the same method as in Chapter II, Figure 19. A29 significantly suppressed the CTL response (p<0.05). SEM was less than 6%. Concentrations of A29 TsF at .1 yg/ml had no significant suppressive effect on the CTL assay.  70-i  60-  1 0.5 0.1 SPLEEN CELL CONCENTRATION/ml(x 1 0  7 )  Figure 35. Effect of panning DBA/2 splenocytes over A29 TsF results in enhanced  CTL responses.  DBA/2 spleen cells were panned over affinity  purified A29 ( 0 ), or BW5147 ( O ) coated plates, and the nonadherent cells were assayed for cytotoxicity of P815 tumor cells. Thereafter, the method used was as described in Chapter II. Cells were 7  titrated in doubling dilutions from 10 /ml. Cells panned over A29 showed significantly enhanced CTL (indicating suppressors had been 7  removed),  p values were <.05 for 1 and .1 x 10 cells per ml. The SEM  was much less than 12% in a l l replicate sets except one.  10?  F i g u r e 36.  Growth o f P815 tumor i n mice r e c e i v i n g u n d i l u t e d A29 TsF  i s o l a t e d from B16G-4B columns.  8 DBA/2 mice r e c e i v e d A29 TsF ( i . v . ) and  4 10  P815 c e l l s ( s . c . ) c o n c u r r e n t l y .  r e c e i v i n g A29 and P815; ( 0---0  ( x  -x ) , growth o f tumor i n mice  ), growth o f P815 i n c o n t r o l mice.  108  Figure 37.  Survival curves for Figure 36.  ( x  x ), survival curve of  4  animals receiving undiluted A29 T3F and 10 ( o  P815 cells simultaneously;  0 ), survival curve of control animals receiving an equivalent  tumor dose.  109  DISCUSSION  The f i r s t experiment reported in this Chapter involved screening of spleen c e l l extracts from normal and tumor-bearing DBA/2 mice for the presence of nominal TsF (all material reactive to B16G), P815  specific  TsFl (the Bl6G-reactive material binding to P815 columns) and anti-idiotypic TsF2 (the Bl6G-reactlve material binding to AlO columns). The results as shown in figure 28 in which l t can be seen that in tumor bearing animals, levels of both antigen binding putative TsFl and anti-idiotypic (or TsFl-binding) TsF2 become elevated shortly after tumor inoculation.  The TsFl levels rapidly rise to a concentration of about 2x  over background and remain at this point throughout tumor growth. More interesting perhaps was the observation that Ts^F material binding to the AlO column increased rapidly to levels 5x over background and maintained at this point until about day 9 after which levels f a l l rapidly.  The meaning of these changes can only be speculated upon at this  time but these kinetics were highly reproducible from one experiment to another (three in total were performed).  These results provided good  evidence for the possible existence of anti-idiotypic TsF2 in this system, but they also indicated that i f a TsF2 was being detected by this procedure, i t shared serological characteristics with the TsFl prototype, AlO, since both reacted with the MAb B16G and with a rabbit antisera raised to AlO. The screening for an anti-idiotypic Ts2 prototype hybridoma involved an ELISA containing limiting amounts of AlO and development with  B16G.  The A29 hybridoma was selected from such a screen of 2,500 hybridomas  110  Figure 38.  The P815 suppressor network.  TsC^ populations (of which AlO  is the hybrid analogue) recognize a specific antigen on the surface of P815, which we have isolated and labelled Ao Ag ( A ). TsC^ populations release a factor TsF^ (AlO TsF) which stimulates id TsCg populations of which A29 is presumably the hybrid analogue. The existence of a TsC^ population has not yet been proven in this systen but is inferred by the kinetics of the suppression (see Discussion). TsC^ populations appear to be the targets of down regulation or contrasuppression (Fig. 28) and may also be their inducer as well, as A29  Ill  TsF administration i n vivo may lead to CS effects (Discussion). "because supernatants from A29 gave rise to enhanced ELISA reactivity in this system, an indication that additional Bl6G-reactive material was present in the wells.  That the product of the A29 hybridoma was truly  anti-idiotypic was established by showing that i t could be affinity purified over columns to which AlO had been linked.  Material eluting from  these columns was reactive with B16G. The possibility that this reactivity was attributable to AlO leaching from the column was discounted on the grounds that when irrelevant TsF's such as BW5147 ascites, the parental line used in the fusions, or Fdll, were passed over the AlO TsF column, acid eluates from i t did not contain appreciable levels of B16G reactive material.  It appeared thus, that A29, while possibly being the  product of a Ts2 prototype, was producing an anti-idiotypic factor which shared biochemical properties with the AlO TsFl prototype.  This was  further established by SDS-PAGE of A29 in which the gel patterns of the A29 factor resemble closely those of AlO. In order to determine whether the soluble products from either A29 or AlO would have a direct effect, at the membrane level on their 2+ counterpart, Ca flux experiments were carried out. 2+ changes in the concentrations of [Ca]  It i s agreed that  i represent signals induced by  cross-linking of cell surface receptors, and can thus be used to evaluate the specific triggering of a c e l l line. to B16G and none to an irrelevant MAb.  Both hybridomas showed reactivity However, in terms of factors, A29  showed responsiveness only to AlO and none to either an unrelated factor, Fdll or A29 i t s e l f .  Similarly, AlO reacted to stimulation with A29  factor but not with Fdll TsF, AlO TsF or polyclonal TsF isolated from DBA  112  spleens over B16G (Chapter I). These results support the contention that there is connectivity between A29 and AlO and the possibility of network interactions. In terms of biological activity, A29 TsF was examined for i t s effect on the generation of P815 specific CTL in the syngeneic system.  The  results showed that A29, at different concentrations, has a significantly suppressive effect on this assay.  In earlier experiments, i t was shown  that AlO had analogous effects on the generation of CTL's.  Whether these  two systems are interdependent (i.e., Tsl -» Ts2 in the normal cell population) has not yet been established, but the end result of adding AlO or A29 to CTL cultures is the same. When attempts were made to determine the effects of A29 factor in vivo, in 4 experiments out of 6, the effect of A29 was to accelerate tumor growth and death, as was observed with AlO TsF.  However, in two experiments we observed in some individuals a  reversed effect, in that some of the experimental animals underwent tumor regression and experienced long term survival.  At this time, i t is  d i f f i c u l t to say whether this is a dose effect, because i t is extremely d i f f i c u l t to obtain definitive protein measurements on affinity purified A29, since yields are very low and we have no way of knowing, with each preparation made by affinity purification, what proportion of the isolated material is s t i l l biologically active.  However, these paradoxical results  do support the possibility that, i f A29 represents a TsF2 transducer, then under some circumstances, i t could trigger contrasuppression and enhanced resistance instead of suppression. The possibility that this is the case  113  is further supported by the observations of Figure 28, in which Ts2 populations are probably the target of down-regulation  after day  7-9.  Because the cells we are working with are hybridomas fused to BW5147, the Ly phenotype of either AlO or A29 would not provide information regarding the precise place of each in suppressor cell circuitry.  We also  do not have appropriate reagents to determine whether the TsF material we are isolating has anti-I-J reactive regions since these lines were derived from H-2^ mice.  Recent data would indicate that antigen binding sites  and I - J molecules from TsC are not covalently linked (130), so concern +  regarding this property may or may not be important.  It is clear that  there is s t i l l considerable work to be done in elucidating suppressor circuitry.  Nonetheless, the results suggest that AlO represents a f i r s t  order suppressor c e l l designated as either Tsl or a T suppressor inducer in terms of the two prevailing models (126,130) and that A29 represents a Ts2 or T suppressor transducer depending on the model. From the results reported here, a number of observations can be made. The A29 hybridoma secretes a molecule which has the following physical properties: i t appears to bind in a specific manner to the AlO molecule which represents a TsFl prototype, i t is bound by the B16G  MAb  thus implying that i t has structural features in common with AlO and other TsF's described raised in this laboratory, and i t s properties on SDS-PAGE 2+ are similar to AlO.  The A29 cell line i t s e l f responds in Ca  experiments in a specific manner to both AlO and B16G.  flux  In terms of i t s  biological properties i t has been shown that i t is capable of suppressing  114  the In vitro generation of CTL specific for P815 and enhancing tumor growth in vivo.  The major characteristic of A29 which suggests its role  a 3 a Ts2 is the anti-idiotypic nature of the molecule secreted by i t . Otherwise i t s properties are similar to those described for AlO.  That  both cell lines and their factors influence the immune response to P815 appears to be the case and therefore network connection between the two is implied since A29 must act via idiotypic interactions because i t does not directly bind antigen. An important consideration which must ultimately be addressed is raised by these data.  Are the suppressor regulators identified as  down-regulating the anti-id Ts^'s in this system restricted to the P815 suppressor circuit, or are they pan-reactive in their effects?  Since only  the TsCg population seems to be the target of "contrasuppression"; this argues that the contrasuppressor (CS) effects observed are idiotypic in nature or directed to a special "CS-induction" sequence on the TsF2.  If  the latter proves to be the case, then injection of A29 Ts2 factor or the "CS-inducing peptide" of the A29 molecule could  3erve  as a general  immunopotentiator useful as a therapy in conditions such as those cancers in which the anti tumor responses are blocked by suppressor circuits. Sequence analysis of AlO and A29 derived TsF's are therefore essential to an understanding of CS and suppressor regulation in the P815  system.  The existence of a Ts3 population in the P8I5 circuit is neither confirmed nor denied by these results, though the kinetics of the system favours i t s existence; note Figure 28 which demonstrates that Ts2 levels are being driven downward prior to (day +10) the time of peak suppression  115  (day +13) indicating the possible existence of a 3rd effector population (Ts3) which had been stimulated by Ts2 prior to the CS down regulation. Only the acquisition of a Ts3 line or hybrid analogue w i l l solve this question unequivocally.  116  Chapter Summary  This Chapter describes the isolation and characterization of a T c e l l v.  hybridoma (A29) which secretes a factor which exhibits anti-idiotypic and immune modulating characteristics.  The A29 cell line is thought to  represent the hybrid analogue of the Ts^ suppressor cell population in the cascade regulating the immune response to the P815 tumor in DBA/2 mice.  This TsF^ molecule is reactive with the monoclonal antibody B16G,  shown previously by us to bind a public specificity of T suppressor factors, and A29 TsF also exhibits specific binding to a TsF^ secreted by another T cell hybridoma, AlO, which shows specificity for antigen from the P815 tumor. A29 i t s e l f does not exhibit binding to P815 antigens. Affinity purified material from A29 appears to share characteristics with AlO molecules in that the predominant material has an apparent molecular weight of 70 kD with breakdown products of about 45 and 25 kD. Studies with calcium flux of A29 cells showed that they respond significantly and specifically upon exposure to AlO TsF stimulus. It was shown further that affinity purified A29 TsF molecules can specifically suppress the in vitro generation of syngeneic CTL to the P815 tumor, and that panning of DBA/2 splenocytes over A29-TsF-coated plates renders cell populations capable of generating a higher in vitro CTL response to P815 than appropriately treated controls.  Therefore, on the basis of these data, A29 f u l f i l l s the  necessary criteria for i t to be designated a hybrid analogue of the aid TsC  in the P815 suppressor network.  117  Chapter IV  Suppressor Deletion Therapy Using Bl6G/Toxln  Conjugates  INTRODUCTION  The monoclonal antibody B16G appears to bind to a constant epitope of T suppressor factors (TsF) produced by DBA/2 mice, CBA mice and humans. The antibody binds to a protein of 70-80 kD molecular weight which may break down into 45 and 25 kD subunits. This molecule, when purified  from  DBA/2 splenocytes, hybridomas or tonsillar tissue is immunosuppressive in the appropriate biological assay.  In panning experiments, B16G was found  to be capable of removing T suppressor cells (TsC), thus establishing that the epitope with which B16G reacts i s also present on the surface of a population of TsC (112). It has been established in our laboratory, as well as in others (4-6) that the growth of P815 mastocytoma cells in syngeneic DBA/2 mice stimulates the development of TsC specific for the tumor and capable of abrogating the development of cytotoxic T lymphocytes (CTL) with specificity for P815, this suppression being mediated by specific TsF's (140).  Chapter II described the isolation of a T cell hybridoma (AlO)  which secretes such a TsF with specificity for the P815 tumor in that i t specifically accelerated P815 tumor growth in vivo and suppressed the generation of P815-specific CTL in vitro (141).  118  In other studies in this laboratory, we have shown that hematoporphyrin  (Hp), conjugated to MAbs directed to tumor associated  antigens can act as an immunotoxin both in vivo and in vitro (142,143). Mice bearing the methylcholanthrene induced tumor (M-I) on their b a c k 3 and treated by an intravenous injection of specific MAb-Hp conjugates, were left in the dark for various time periods, following which they were exposed to light.  Animals which were left under dark conditions for 5  days prior to light exposure experienced tumor regression In about 50% of treated mice (142) whereas a l l controls developed progressively growing tumors and ultimately died.  Similarly, MAb-Hp conjugates with specificity  for a human leukemia-associated antigen (CAMAL) were shown to be capable of specifically k i l l i n g antigen bearing c e l l lines after laser activation of treated cells (143). The present study was undertaken to determine whether B16G-Hp conjugates could k i l l TsC in vitro and in vivo and whether such treatment would influence the growth of the P815 tumor in DBA/2 mice.  *.  119  MATERIALS AND METHODS  Hematoporphyrin.  Hematoporphyrin dihydrochloride (95%) was obtained from  Sigma Chemical Co., St. Louis, MO.  Hematoporphyrin conjugation.  (Figure 39).  The conjugation procedure was developed and  performed by Dr. Lily Chi-Kit Wat, Department of Botany, University of B.C.  Briefly, 20 mg of Hp-2 HC1 in 1.25 ml HgO and .8 ml  N-N-dimethylformamide was added to 20 mg l-ethyl-3-(3-dimethyl aminopropyl carbodiimide HC1) in .6 ml tt^O. 15 mg of antibody to be conjugated added in 5 ml H^O and allowed to bind for 5 hrs at pH 6.7.  was  The reaction  was stopped by the addition of 50 y l monoethanolamide and allowed to stand overnight.  The conjugate was dialysed exhaustively against .001 M  phosphate buffer pH and against one change of PBS. by passage through a G25 column.  Unbound HP w a 3 removed  The general method described above is  known as an EDCI coupling reaction.  Spectrophotometry assay.  Amount of bound HP was determined  photometrically by absorption analysis at 505nm (.644  0D = 80 yg  Hp/ml). Protein concentration was determined by absorption analysis at 280nm (1.400 = 1.0 mg/ml). Any cross absorbance of HP at 280nm is corrected for in these calculations.  120  Level3  of Hematoprophyrin Labeling.  A f f i n i t y p u r i f i e d rabbit anti-mouse  Ig (RaMIg), p u r i f i e d B16G, and a p u r i f i e d i r r e l e v a n t MAb (C-MAb) of the same subclass as B16G were labeled with hematoporphyrin (Hp) using the carbodiimide reaction.  The r e l a t i v e levels of labeling were as follows:  RaMIg Hp, 110 yg Hp/mg RaMIg; B16G-Hp, 156 jig Hp/mg B16G; C-MAb-Hp, 95yg Hp/mg C-MAb (control monoclonal).  The conjugated  antibodies were tested f o r a c t i v i t y by ELISA, and conjugated Hp was quantified spectrophotometrically as described above.  In vivo Experiments.  DBA/2 mice were injected subcutaneously i n the right  flank with 10^ P815 c e l l s i n 200 jil PBS. When this tumor dose i s used, tumors usually become palpable 6-7 days a f t e r i n j e c t i o n and death occurs within 15 to 30 days.  On day 8, groups of tumor bearing animals,  selected ramdomly, were injected intravenously with 150 y l of PBS containing either nothing, or B16G-Hp conjugate, or Hp, or B16G plus Hp i n an unconjugated form. alone or C-MAb-Hp also.  Some experiments included groups injected with B16G A l l animals were maintained i n the dark f o r 2 h  after which they were exposed to a strong incandescent l i g h t (22.5 2 mW/cm  24 cm above the animals) f o r a further 5 h. Thereafter, they  were maintained under normal conditions.  Minimal e f f e c t i v e levels of  B16G-Hp were determined with preliminary experiments.  The levels of Hp i n  a l l groups i n the f i n a l d e f i n i t i v e experiments were adjusted so that a l l animals received 50 yg of Hp (approximately 2.5 mg/kg). examined d a i l y f o r tumor growth and s u r v i v a l .  Animals were  Tumor size was assessed  with calipers using two cross-sectional measurements to give an estimate  121  of the area of the tumor. A total of 5 different experiments of t h i 3 nature were carried out. Ik  In vitro k i l l i n g with Hp conjugates.  Cells to be used for in vitro  k i l l i n g were harvested and washed in DME plus HEPES. When direct k i l l i n g 5  with B16G-Hp was assessed, experiments were done as follows. 5x10 cells were suspended in 1.0 ml of DME/HEPES containing either B16G-Hp or C-MAb-Hp at Hp concentrations of 320, 160, 80, 40 and 20 ng Hp/ml.  Cells  were incubated in the dark at 37°C for 1 h following which they were washed 3 times in 5.0 ml volumes of DME/HEPES and finally suspended in 1.0 ml of DME/HEPES. At this point (time 0), three 100 pi aliquots of each preparation were dispensed into flat bottom microtitre wells. The remainder of the c e l l suspensions (700 pi) were exposed to strong light 2 (22.5 mW/cm ) at a distance of 20 cm for 1 h, after which a further three 100 pi aliquots were dispensed to three microtitre wells. H -thymidine (NEN) diluted in DME/HEPES containing 20% FCS was then added to a l l microtitre wells in 100 pi aliquots so that 2 pCi of 3  3  H-thymidine was added to each well. Cultures were incubated for 18 h at 37°C in a humidified 10% C0 incubator after which they were 2  3  harvested on a MASH harvester, and H-thymidine incorporation measured with a Packard scintillation counter (TRI-CARB Model 4550). When indirect k i l l i n g of AlO cells with B16G was assessed using RaMIg-Hp, the AlO cells to be used were prepared as described above. Cells were then exposed'to 50 pg/ml of either B16G or C-MAb at 4° for 30 min after which they were washed in DME/HEPES. Aliquots of each c e l l  122  population were then exposed for a further 30 min at 4°C in the dark to varying concentrations of RaMIg.Hp between 2.0 ug/ml and 1 5 ng/ml of Hp.  Controls of unlabeled MAbs and RaMIg were also run.  Cells were 3  washed three times, exposed to light, and cultured with described above.  H-thymidine as  123  Figure 39.  S t r u c t u r e o f the hematoporphyriri  molecule  124  RESULTS  In vitro experiments were run to determine whether B16G-Hp conjugates were capable of k i l l i n g the AlO hybridoma, considered to represent the immortalized analogue of P815-specific TsC.  Cells from a number of c e l l  lines were incubated with various concentrations of B16G-Hp or Hp alone, washed, resuspended, exposed to light for 2 hours and incubated overnight.  Percent k i l l was assessed by comparison to cells treated as  the experimental samples but not exposed to either B16G-Hp or Hp. The results (Fig. 40)  3how  that B16G-Hp effectively k i l l s essentially a l l of  the AlO cells at doses of 75 ng HP whereas c e l l k i l l i n g with Hp alone, or with Hp coupled to an irrelevant antibody have no effect at this dose. P815 tumor cells were found to be completely resistant to B16G-HP k i l l i n g even at doses of 75 ng HP-B16G, thus demonstrating that direct tumor k i l l i n g was not responsible for the results seen in vivo. The tumor line L1210 proved slightly susceptible to k i l l i n g by B16G-HP, possibly because as a transformed T-cell, i t bore antigens to which B16G can bind. No other tumor line tested (most importantly, P815), as well as a B-cell hybridoma (C-613) was significantly affected by B16G-HP at these doses. In additional in vitro studies, unlabelled B16G or control Ab was used as the f i r s t layer in a double binding system. to k i l l cells labelled with mouse Ig.  RaMIg-HP was used  Only B16G treatment rendered AlO  cells susceptible to RaMIg-HP k i l l i n g (Fig. 41) at doses up to 125 ng HP-RaMTg; no toxicity towards AlO cells "labelled" with control Ab was observed until levels over 250 ng HP-RaMTg were reached; at these  v.  Figure 40.  The In vitro effect of BI6G-Hp in comparison to that of  C-MAb-Hp on AlO ( • ), L1210 ( 0 ), or P815 ( • ). Percent death 3  was calculated from the level of Inhibition of  H-thymldine  Incorporation of treated cells in comparison to control samples of cultures taken at time zero and maintained in the dark during culture  ng HPIcu R A M I G - H P I  Figure 41.  The effect of RaMIg-Hp on AlO cells in vitro previously  labeled with either B16G ( • ) or C-MAb ( 0 ). estimated as described for figure 3.  Percent death was  127  levels, the k i l l i n g is non-specific.  These results were found to be  reproducible and indicated the feasibility of in vivo manipulation of T cell subsets. For In vivo studies, Female DBA/2 mice of 6-8 weeks of age were 4  injected subcutaneously in the right flank with 10 described (136).  P815 cells as  When this cell do3e was used, tumors became palpable 6-7  days after injection with death occurring between day 15 and 30. Spontaneous regression was not observed at this dose.  Animals were  injected intravenously on day 8 with B16G-Hp, B16G + Hp, Hp alone, B16G or PBS, under minimal light conditions.  Animals were kept in the dark for 2  hours after which they were exposed to bright fluorescent light (1.0 2 mW/cm 24 cm above animals) for a further 5 hours. maintained under normal conditions.  Thereafter they were  Animals were examined daily for tumor  growth, and survival. Tumors were measured by calipers using two cross-sectional measurements to give an estimate of the area of the tumor. The survival curves of one such experiment are shown in Fig. 42 in which i t can be seen that whereas a l l control animals in both groups were dead by day 30, less than half the B16G-Hp treated animals were, and 3 of 8 of these animals ultimately demonstrated complete regression of their tumors.  Growth curves from the same experiment (Fig. 43) reflect this  result.  Animals receiving B16G-HP therapy grow their tumors at less than  1/2 the rate of control mice. B16G-Hp have been run to date.  A total of 5 in vivo experiments with A summary of survival in these experiments  is shown in Table III. In a l l experiments, approximately 1/3 of the specifically treated mice underwent complete tumor regression, whereas  128  DAYS  »  Figure 42. Survival curves of P815 bearing DBA/2 mice. 4  DBA/2 mice were  injected with 10 viable P815 cells subcutaneously on day 0. On day 8, when tumors were uniformly palpable, the mice were randomized into control and test groups.  Each group (8 mice) w a 3 Injected intravenously with free  Hp (50 pg) plus B16G (100 yg), PBS or B16G-Hp conjugate. levels in each group were identical.  Hp and MAb  A l l mice were rested in the dark for  3 h after injection, and were the exposed to bright Incandescent light for 5 hours.  Thereafter, animals were maintained under normal conditions.  Mice were observed dally for survival.  (0  -0 ), animals receiving PBS;  ( O • ), animals receiving unconjugated B16G plus Hp; ( x animals receiving B16G-Hp conjugate.  x ),  129  Figure 43. Growth of P815 tumor in mice receiving B16G-Hp treatment. Animals were treated as described above with B16G-Hp ( x ( A  A ), B16G and Hp in an uncoupled form ( • •  mock injection ( A P815 cells s.c.  x ), PBS alone  • ), or PBS 4  A ) seven days after a lethal injection of 10  130  none of the control animals did.  A further analysis of the B16G-Hp  treated animals was undertaken. The pattern of tumor growth in mice undergoing regression was compared with those treated animals which eventually died of tumors. The results (Fig. 44)  3how  that while tumor  size in regressor mice on days 10 and 11 was not significantly less than that of the progressor mice, by day 13 the differences were highly significant (p<.005).  By day 18 about 1/3 of regressor mice had  completely rejected their tumors whereas the remainder showed scabbing at the tumor  3 i t e .  v.  131  tO  11  12  13 DAYS  POST  14 TUMOR  INJECTION  15  16  17  •  Figure 44. Comparison of tumor growth In progressor (ultimately died of 4 tumor) and regressor*(ultimately cured) DBA/2 mice receiving 10 P815 cells and B16G-HP 8 days later.  Error bars for each group are shown. By  day 13, differences are highly significant between these two groups. ( o 0 ), survival curve of progressor mice; ( x x ), curve of regressor mice.  132  DISCUSSION  In a study done nearly a decade ago in this laboratory, i t was shown that during the growth of P815 tumors, P815-specific CTL develop at the early stages, but are subsequently overwhelmed by the development of a supressor population (144).  A period of temporary regression of tumors i s  frequently seen in P815 bearing mice during the time that CTL's dominate (days 11-14 after tumor injection).  It would appear that Bl6G-Hp and  exposure to light, may be an effective mechanism for eliminating TsC in these animals so that the CTL's can eliminate tumors.  Hp h a 3 been used as  a chemotherapeutic agent for the treatment of a variety of cancers in humans (145,146) since i t has been found to have a slightly greater affinity for malignant tissue than i t does for normal.  We have shown that  Hp-MAb conjugates can be used effectively as immunotoxins (142,143) with MAb's with specificity for tumor associated antigens.  In this study, we  show that Hp-MAb conjugates can be used to eliminate selectively subpopulations of lymphoid cells in the circulation; presumably sufficient light penetrates the skin of treated animals (in the t a i l and ears) to effectively activate the Hp which mediates cell k i l l i n g .  Animals treated  in this way did not exhibit any toxic side effects as a result of either Hp or as a result of selective deletion of TsC.  By way of contrast,  Dougherty has reported mortality rates of 50% in mice receiving the high doses of HP (5 yg HP/Kg body wt) required to effect tumor therapy (147) in an unconjugated form.  Though i t appeared unlikely that these effects  were attributable to causes other than immune  133  modulation, (since they were not seen in any of the control groups receiving Hp in various forms) i t was recognized that one alternate explanation for the regressions was the possibility that B16G-Hp was having an effect directly on P815 cells.  Although this seemed unlikely in  light of the fact that Hp alone, C-MAb-Hp, or B16G plus Hp had no effect on the course of tumor growth, studies were underaken to examine the effect of B16G-Hp directly on P815 cells and on those of the AlO c e l l line, which is an immortalized TsC hybridoma, and secretes material with which B16G reacts. The results, some of which are presented i n Figures 40 and 41, show that Bl6G-Hp or B16G followed by RoMIg-Hp are selectively cytotoxic to AlO cells at the levels shown in this study and have no specific cytotoxicity for P815 cells.  Another argument against the  possibility that B16G-Hp is having an effect directly on P815 cells is the actual timing of the experiments done. B16G-Hp was administered i.v. only 2 h prior to light exposure.  Under these conditions, most of the labelled  material would s t i l l be in the circulation since insufficient time would have elapsed for accumulation of the material in other parts of the body, (i.e., tumor cells) thus arguing for i t s effect being attributable to i t s binding to circulating cells. As far as the possible consequences of TsC deletion the obvious danger here is the possible induction of anti-self immune reactions because of the elimination of self tolerance maintaining TsC populations. A study carried out by Nossal et a l . (148) has demonstrated that this may not be a concern; clonal deletion or silencing (anergy) of self-reactive subsets appears to be the dominant mechanism of maintenance of tolerance to self.  134  Table IV.  Summary of B16G-Hp Experiments.  This table summarizes 5 4  experiments in which a l l 137 animals received 10 P815 cells subcutaneously.  38 animals were treated on day 8 with B16G-Hp  and 13 of these underwent complete tumor regression. No controls receiving B16G and Hp, free Hp, B16G or PBS survived longer than 50 days (average survival time in a l l control groups was 22 days). Treatment  I.  Number of  Number of animals  %  animals  surviving  survival  tested  >100 days  Untreated (PBS)  48  0  0  B16G + Hp  26  0  0  Hp  17  0  0  8  0  0  99  0  Q%_  3  33  Controls  B16G Total II.  B16G-Hp Experiment 1  9  Experiment 2  8  3  38  Experiment 3  8  3  38  2  40  8  2  25  38  13  34%  Experiment 4 Experiment 5 Total  5  135  In one experiment, vigorous TsC deletion using anti I-J antiserum failed to break tolerance to alloantigens to which the recipient animals were neonatally tolerized (148). These results are preliminary, and further in depth studies in our lab are ongoing at this time.  Since B16G has been shown by us to bind to  a suppressive molecule isolated from human tonsils (Chapter I, and (ref. 121)) as well as to the murine TsF's, and i t has also been shown to bind to a subpopulation of human lymphoid cells, as assessed by immunofluorescence.  Thus, the technique of suppressor deletion therapy  may be of significance in the management of human cancers.  136  Chapter Summary  The monoclonal antibody B16G (which recognizes a constant epitope on T suppressor cells and their soluble factors in DBA/2 mice) has been described in previous Chapters.  In this study, we show that when this  monoclonal antibody was covalently linked to the photoactivable molecule hematoporphyrin, and injected intravenously into P815 tumor bearing mice which were subsequently exposed to light, tumors underwent permanent regression in 20-40% of these mice.  A l l control animals died within an  average of 20-22 days after tumor c e l l injection.  It i s suggested that  tumor regression is attributable to immune mechanisms facilitated by the elimination of a population of T suppressor cells.  Chapter V  The Therapeutic Potential of Purified Monoclonal TsF as an Anti-Tumor Vaccine  INTRODUCTION  Attempts to boost tumor immunity by vaccination therapy have been numerous over the past 100  year3.  Generally, these attempts f a l l into  three categories; attempts to immunize a recipient using tumor cells as the immunogen, attempts to immunize using tumor "specific" antigen, and attempts using irrelevant antigens such as BCG,  in an effort to increase  immunity in a non-specific manner. As indicated in the Thesis Introduction, a l l of these attempts have largely failed, possibly because increased immune responses to tumor cells ("self") are down-regulated strongly by suppressor c e l l populations. In this study, a new approach was used.  The ability of the  tumor-specific T suppressor factor (TsF) isolated from the T cell hybridoma, AlO, to act as an immunogen in DBA/2 mice was investigated. The TsF was affinity purified from ascites over an immunoadsorbent column containing the B16G monoclonal which has specificity for the TsF molecule, or over columns containing membrane extracts of the P815 mastocytoma (containing the tumor antigen for which AlO is specific).  The specificity  control was BW5147 (the fusion partner for AlO) membrane extracts treated in the same way as AlO.  DBA/2 mice were immunized with the affinity  138  purified material or PBS and subsequently challenged with either the  P815  tumor or the L1210 DBA/2 thymoma. When mice were immunized with material affinity purified over B16G,  eluted material from both AlO ascites and  BW5147 membrane extracts enhanced resistance to both P815 and  L1210  challenge indicating that B16G was binding immunogenic material derived from both preparations which exerted a tumor-protective  effect.  However,  when a P815 affinity column was used, protective material was eluted only from AlO ascites, and this bestowed resistance to both P815 and L1210. When irradiated whole cells were used as immunogens, only AlO cells stimulated anti-tumor immunity and this appeared to be directed specifically to the P815 tumor. The implications of these findings in terms of the potential for immune modulation with anti-suppressor therapy, and the specificity of the B16G monoclonal, are discussed.  The  demonstration of B16G binding material (TsF) in the membranes (but not the ascites) of the BW5147 line is also of significance to investigators using BW5147 fused suppressor hybridomas.  139  MATERIALS AND METHODS  Experimental animals.  DBA/2 female mice between 6-8 weeks of age were '  used for a l l assays of P815 and L1210 tumor growth.  CBAxBalb/c P mice 1  were used to grow AlO T-cell hybridomas as an ascitic tumor as described previously (141). A l l animals  U 3 e d  C57B1/6 mice were used for growth of the EL4 tumor. in these experiments were raised in the animal breeding  f a c i l i t y of the Microbiology department at t h i 3  Cell lines.  University.  The DBA/2 tumor mastocytoma line P815 and the L1210 T c e l l  leukaemia line, the AKR thymoma BW5147, and the EL4 c e l l line from C57B1/6 have been maintained in this laboratory for 12 years and were originally obtained from ATCC. The T-cell hybrid line AlO and the B16G B-cell hybridoma were raised and maintained in this laboratory as previously described (112,141). g BW5147 membrane extracts.  10 BW5147 cells in log phase were lysed with  5 ml lysis buffer (.14 M NaCl, 1.5 mM MgClg, 10 mM Tris-HCl, 0.1% NP40) and centrifuged at 11,500 rpm for 30 minutes to remove non-solubillzed material.  The c e l l lysate was dialysed against PBS at 4°C to remove  detergent prior to immunoadsorbence over P815-Sepharose or Bl6G-Sepharose columns as described below.  140  Immunoad3orbent3.  AlO TsF was purified from ascitic fluid over B16G  immunoadsorbent columns as previously described in Chapter II. AlO TsF was also purified over P815 membrane extracted antigen attached to Sepharose 4B for some experiments by the method described above.  In both  cases, yields of TsF obtained were equal as previously described. Control preparations (BW5147 membrane extracts) were treated in the same manner.  Immunization with TsF.  DBA/2 mice of 6 weeks of age received a  subcutaneous injection of 20 yg of affinity purified AlO in a 50% emulsion of Complete Freund's adjuvant (CFA) 28 days prior to tumor injection (day -28) and were boosted with an equivalent dose on day -14. AlO-immunized animals were then injected subcutaneously with 10 P815 cells on day 0, in the flank, at a site far removed from the A10/CFA granuloma  (to minimize non-specific anti-tumor effects caused by BCG).  Control animals were immunized on day -28 and -14 with PBS/CFA or BW5147 (fusion partner for AlO hybridoma) ascites "purified" over P815 or B16G affinity columns) in 50% CFA.  Cellular immunizations.  The cell  grown up in DME plus 10% FCS. 2000 rads.  l i n e 3  AlO, BW5147, EL4 and P815 were  Cells were harvested and irradiated with  Cell suspensions in PBS and 50% CFA were administered by  7 subcutaneous injection at a concentration of 10 cells in 200 y l to either DBA/2 or C57B1/6 mice,  animals were rested for two weeks and again  immunized as described. This procedure was repeated after which mice were rested for a further two weeks and challenged with either P815 (3x10  3  141  cells) or L1210 ( l t r cells) for DBA/2 mice and EL4 (10 cells) for C57B1/6 mice.  ELISA.  Animals were monitored daily for tumor growth and survival.  DBA/2 or Balb/c mice were immunized and boosted with AlO TsF as  described above.  On day 28, 3 mice from each strain were bled, and their  sera tested for reactivity to AlO coated or irrelevant antigen (irrelevant hybridoima ascites eluted from B16G columns) coated ELISA plates. mouse serum served a 3 a control.  Normal  Dilutions of sera ranging from 1:100 to  1:3000 were applied in sextuplicate, allowed to bind for 1 h at 37°C, and unbound material was washed away with 3 washes of PBS-tween. Plates were then developed with rabbit anti-mouse Ig conjugated to alkaline phosphatase and Sigma substrate as previously described (120,121,141). Plates were read at 30 min and 1 hr on a Flow Titretek at 405 nm.  Statistical analyses.  Tumor measurement on experimental animals were made  daily after tumors become palpable (day +7) as described (141).  Area  measurements of the tumor size in the AlO groups were compared with control values by Student's t-test. considered significant.  Differences with p values <.05 were  Survival of each group were also followed, and  the same statistical scrutiny applied to these results.  142  RESULTS  DBA/2 mice were Immunized with material eluted from P815 membrane affinity columns, derived from either the AlO T c e l l hybridoma or the 4  BW5147 cell line.  They were subsequently challenged with 10 P815  cells, and monitored for the appearance and growth of palpable tumors. The results (Fig. 45) show that the rate of tumor growth in AlO TsF immunized mice is significantly slower than that observed in animals receiving equivalent material from BW5147 membrane extracts or PBS/CFA. The slowed tumor growth observed was reflected in a significantly prolonged survival rate (Table IV, experiment 4). The specificity of the P815 resistance initiated by active immunization with AlO T3F purified over P815 columns was tested by 4 challenging immunized mice with 10 L1210 tumor cells.  The results  (Fig. 46) show that AlO-immunized mice demonstrated increased resistance to this tumor also, as reflected in slowed tumor growth, a high incidence of tumor rejection, and increased survival time (Table IV, experiment 5). These results indicate that priming animals with the TsF material derived from the AlO c e l l line which bound specifically to the tumor antigen in P815 membranes, resulted in an immune response (presumably anti-suppressor) which renders mice resistant not only to the P815 tumor but also to other tumor c e l l lines.  140  i  7  8  9  »  « DAYS  U  12 POST  TUMOR  M INJECTION  U  16  17  18  •  F i g u r e 45. Growth o f P815 tumor i n DBA/2 mice p r e v i o u s l y immunized w i t h m a t e r i a l from e i t h e r BU5147 membrane e x t r a c t s o r AlO a s c i t e s e l u t e d from a f f i n i t y columns c o n t a i n i n g membrane e x t r a c t s o f P815 tumor c e l l s .  Mice  were immunized t w i c e 28 and 14 days p r i o r t o tumor c h a l l e n g e w i t h  antigen  o r PBS i n 50% CFA.  ( 0 — - 0 ) , PBS/CFA c o n t r o l ; ( a  derived material; ( x  x ) , AlO d e r i v e d m a t e r i a l .  a ) , BW5147  144  Figure 46. Growth o f L1210 tumor i n DBA/2 mice p r e v i o u s l y immunized  with  m a t e r i a l from e i t h e r BW5147 membrane e x t r a c t s o r AlO a s c i t e s e l u t e d f r o m a f f i n i t y columns c o n t a i n i n g membrane e x t r a c t s o f P815 tumor c e l l s .  Mice  were immunized t w i c e , 28 and 14 days p r i o r t o tumor c e l l c h a l l e n g e ,  with  a n t i g e n o r PBS i n 50% CFA. derived m a t e r i a l ; ( x  ( 0---0 ). PBS/CFA; ( Q — a ), BW5147  x ) , AlO d e r i v e d m a t e r i a l .  145  Since the AlO T c e l l hybridoma was isolated originally by screening for cells producing materials reactive to the MAb B16G (141) which reacts with an invariant epitope on murine T3F's (112,120) further experiments were carrried out to determine whether potentially protective immunogenic materials could be derived from AlO ascites affinity purified over B16G columns.  In these experiments, BW5147 membrane extracts were used as  control material. 4 10  P815 cells.  Mice were immunized as described and challenged with  A typical set of results is shown in figure 47.  It can  be seen that when materials afffinity purified over B16G columns are used as immunogens, increased tumor resistance over PBS/CFA controls i s apparent in mice immunized with both AlO and BW5147 derived material. When mice immunized with AlO or BW5147 derived material were challenged with L1210  cells, they also exhibited marked resistance to challenge, in  that over half of them completely rejected the tumor c e l l challenge (figure 48 and Table IV, experiment 6).  A summary of the various  experiments done using B16G- or P815-affinity purified materials as immunogens is shown in Table IV, in which i t can be seen that a l l experiments done showed the same results as those described here.  The use  of BW5147 membrane extracts as a control may seem somewhat surprising, but has come about from the following observations.  BW5147 ascites has no  B16G reactive material, as verified by elution from B16G-4B columns and testing in the ELISA (Chapter II). Similarly, BW ascites "purified" over B16G and used to immunize mice has no effect on tumor growth (Fig. 49). However, BW5147 apparently contains B16G reactive material in i t s membrane, a fact that was discovered when comparative tests to determine  146  200-  7  8  9  10  11 DAYS  12 POST  TUMOR  13  14  INJECTION  15  16  17  18  •  Figure 47. Growth of P815 tumor in DBA/2 mice previously immunized with material from either BW5147 membrane extracts or AlO ascites eluted from B16G affinity columns. Mice were immunized twice, 28 and 14 days prior to tumor cell challenge, with eluted antigen (TsF) or PBS in 50% CFA. ( 0 — 0 ), PBS/CFA control; ( • ( x  x ), AlO derived material.  • ), BW5147 derived material;  147  whether better yields of TsF could be obtained from AlO c e l l lysates or ascites could be obtained were run.  BW5147 cells were used as a control,  and surprisingly, their membrane extracts were discovered to be B16G reactive.  Subsequently, immunization with BW5147 cells was found to be  effective (below).  BW5147 has been hypothesized to be of suppressor  origin (but non-secreting) and is demonstrably suppressive in some assays though no antigen specificity can be assigned (144) and in this context, the results make sense. A further study was undertaken in which mice were immunized with irradiated cells from various lines (AlO, P815, BW5147, EL4). mice were challenged with P815, L1210 or EL4.  Immunized  The results (Table V) show  clearly that this protocol enhances tumor resistance only when AlO cells are used to prime animals, and that the resistance is directed only to the P815 tumor system, indicating that when whole cells are used, as opposed to secreted factors, the response i s anti-idiotypic in that i t is specific only for the P815 tumor antigen against which AlO reacts. In order to determine whether experimental animals immunized with AlO TsF generated specific antibody reactive with the AlO molecule, immunized animals (DBA/2 and Balb/c) were bled and their sera tested in ELISA for reactivity to AlO.  The results (fig. 50) show that antibody specific for  AlO does develop in both strains, a result somewhat surprising in light of the fact that the AlO molecule is derived from a DBA/2 parent c e l l and thus, this is a form of autoimmunity.  142  2TO  T5C  TUMOR AREA  50i  9  K>  11 DAYS  13  tt POST  TUMOR  U  INJECTION  15  16  17  U  »  F i g u r e 48. Growth o f L1210 tumor i n DBA/2 mice p r e v i o u s l y  immunized  with  m a t e r i a l from e i t h e r BW5147 membrane e x t r a c t s o r AlO a s c i t e s e l u t e d f r o m 8 1 6 G - a f f i n i t y columns. tumor c e l l c h a l l e n g e ,  Mice were immunized t w i c e , 28 and 14 days p r i o r t o  with eluted antigen  ( 0---0 ), PBS/CFA c o n t r o l ; ' ( a ( x  x ), AlO d e r i v e d  material.  (TsF) o r PBS i n 50% CFA.  • ) , BW5147 d e r i v e d  material;  D A Y S POST 112lO INJECTION  Figure 49.  ,  Survival curves of L1210 tumor bearing mice immunized with:  AlO or BW5147 ascites purified over B16G-4B columns. surival of PBS/CFA mice; ( 0 treated mice; ( x — — x  (•  • ),  0 ), survival of BW5147 B16G eluate/CFA  ), survival of AlO TsF/CFA treated mice.  these mice ultimately exhibited total tumor regression.  25% of  150  BALB/c  DBA/2  Figure 50. ELISA results from sera of Balb/c and DBA/2 mice immunized with affinity-purified AlO. Immunized mice were bled 7 days following a secondary antigenic stimulus with AlO. Three immunized mice from each strain were chosen randomly and bled.  Serum concentrations shown here  were at 1:400 and ELISA readings for 405 run absorbing materials were made at 30 min. ( • ), anti-AlO activity of immune serum on AlO-coated ELISA plates; ( • ), background levels of Immune sera at 1:400 on plates coated with irrelevant antigen; ( 13 ), ELISA reactivity of non-Immune serum at 1:400 on AlO coated plates.  Table V. Effect on tumor growth of active immunization with affinity purified AlO and control materials Expt.  Inrmunoadsorbent used  B16G  Imraunogen used  PBS/CFA  Number of animals  Tumor challenge  Survival time (days ± SEM  p*  Long term survivors ( 100 days free of tumor)  8  10 P815  18.3 ± 1.2  6  10 P815  23.9 ± 2.9  .05  0  .025**  1  0  B16G  BW5147  B16G  AlO  6  10 P815  24.4 ± 2.0  B16G  PBS/CFA  7  5x10 P815  34.14 ± 4.5  B16G  AlO  8  5xl0 P815  32.0 ± 2.7  B16G  PBS/CFA  7  10 P815  25.8 ± 2.9  B16G  BW5147  7  10 P815  32.0 ± 1.4  .05  0  B16G  AlO  9  10 P815  32.6 ± 1.3  .05**  0  P815  PBS/CFA  9  10 P815  29.3 ± 2.6  P815  BW5147  8  10 P815  29.4 ± 2.9  N.S.  0  P815  AlO  7  10 P815  39.6 ± 5.6  .05  0  4  4  3  4  4  4  4  0 N.S.**  4 0  0  Table V (continued) Expt.  5  6  Immunoadsorbent used  Immunogen used  Number of animals  Tumor challenge  Survival time (days ± SEM  10  10 L1210  15.7 ± 0.62  p*  Long term survivors ( 100 days free of tumor)  P815  PBS/CFA  P815  BW5147  9  10 L1210  17.8 ± 1.4  N.S.  0  P815  AlO  9  10 L1210  29.9 ± 4.9  .005**  1  B16G  PBS/CFA  8  10 L1210  18.1 ± 1.1  B16G  BW5147  8  10 L1210  21.0 ± 0  N.S.II  6  B16G  AlO  6  10 L1210  26.0 ± 1.3  .05**  3  4  4  4  4  4  4  0  0  *  p values based on comparison of survival times of treated mice with PBS controls by Student's t-test.  ** p values for these groups were determined on mice dying with tumor.  In groups where complete  regressions in treated mice reached 50% (experiments 2 and 6), p values did not show significance for this reason.  Table VI. Effect on tumor growth of active immunization with irradiated cells in CFA Expt.  Iraraunogen  1  P815  DBA/2  EL4  2  3  Survival time  P  P815  21.1 ± 1.1  N.S.  DBA/2  P815  20.38 ± 2.8  N.S.  BW5147  DBA/2  P815  21.43 ± 2.9  N.S.  AlO  DBA/ 2  P815  30.1 ± 4.3  PBS  DBA/2  P815  19.1 ± 0.6  -  EL4  C57/B1/6  EL4  31.0 ± 1.3  N.S.  BW5147  C57/B1/6  EL4  30.4 ± 1.7  N.S.  AlO  C57/B1/6  EL4  34.2 ± 2.6  N.S.  PBS  C57/B1/6  EL4  33.5 ± 1.8  -  AlO  DBA/2  P815  30.4 ± 3.1  AlO  DBA/2  L1210  18.1 ± 0.74  PBS  DBA/2  P815  25.2 ± 0.8  -  PBS  DBA/2  L1210  18.1 ± 0.9  •-  Mouse strain  Challenge  0.01  0.05 N.S.  r  154  DISCUSSION  In Chapter II, i t was demonstrated that the T cell hybridoma AlO secreted material which could specifically inhibit the immune response to, and thus enhance the growth of the P815 tumor in DBA/2 mice.  Using as a  probe a MAb with specificity for murine TsF's we were able to affinity purify the AlO product which has an apparent molecular weight of 70-80 kD and breaks down to subunits of approximately 45 and 25 KD.  The AlO  product thus appears to f u l f i l l a number of the criteria for an antigen-specific f i r s t order TsF (Tsl).  The present study was undertaken  to determine whether active immunization with the AlO molecule could modulate the resistance of DBA/2 mice to subsequent challenge with P815. The results reported herein show that this kind of protocol significantly enhances resistance to tumor challenge, not only to P815 but also to the unrelated tumor L1210. Since i t was established, some years ago, that the P815 and L1210 tumor do not share suppressor epitopes (137), these results indicate that the AlO-immunized mice are responding not just to the idiotypic regions on the TsF, but also to invariant portions of the molecule, and that this response renders mice capable of suppressing the suppression generated after tumor cell challenge. Since the AlO molecule is derived from the DBA/2 genotype, this observation is somewhat surprising, and can be explained by at least two possible mechanisms. (1)  Presentation of the AlO molecule in CFA renders i t immunogenic at  regions other than the idiotype, thereby effecting general anti-TsF immunity or (2), that TsF molecules in general may stimulate not only an  155  anti-idiotypic response but may also trigger amplification of contrasuppressive elements which respond to non-idiotypic regions of the molecule. The somewhat anomalous observation that immunization of mice with AlO cells rather than the affinity purified factor resulted in the development of enhanced resistance to P815 only indicates that when the AlO molecule is presented as a membrane bound molecule which presents primarily the idiotypic regions of the TsF to stimulate the response, which then proceeds primarily in an antigen specific manner. It is not known at this time whether the resistance to tumor as a result of AlO immunization is mediated exclusively by B-cell or T-cell mechanisms or whether i t is the result of a combination of the two. We know that animals immunized with either the AlO molecule or with irradiated cells develop antibody specific for AlO and that this can be detected by standard ELISA (figure 50).  We also know, from earlier  studies, that antiserum taken from mice immunized with polyclonal P815 affinity purified TsF afforded passive recipients enhanced resistance to challenge with P815 (140). Thus, i t i s likely that the resistance bestowed by AlO immunization is probably at least partially mediated by antibody.  The role of anti-idiotypic or anti-TsF T cell populations  remains to be elucidated; the presence of anti-idiotypic Ts2 cell populations and possibly contrasuppressor effects have been noted in mice pretreated with AlO TsF.  Either must be regarded as breaking tolerance to  a "self" protein the TsF molecule, by rendering i t immunogenic in CFA. Thus, a successful anti-tumor response can be interpreted as a form of auto-immunity.  156  Possibly the most surprising observation made in these experiments was that when material to be used for immunization was purified over B16G affinity columns rather than P815 membrane columns, both AlO and BW5147membrane derived material afforded enhanced tumor resistance.  These  findings were unexpected since BW5147 ascites has been used previously by us as a negative control for the characterization of AlO material, and does not contain isolable Bl6G-reactive material (TsF's) as assayed by ELISA (141); nor do BW5147 ascites Bl6G-Sepharose eluates afford significant protection against P815 tumor growth when used as an immunogen (Fig. 49).  There is evidence that the BW5147 c e l l line has non-specific  suppressor activity in some assays, though no antigen specificity has yet been assigned (127); therefore i t is possible that some product, presumably distinct from the AlO molecule, but sharing an epitope common to TsF's and reactive with B16G, cells.  is present on the surface of BW5147  It is also possible, following this line of reasoning, that this  product, as well as reacting specifically with the B16G MAb, could also stimulate an active anti-suppressor c e l l response when used as an immunogen. Unlike the AlO hybridoma, however, any BW5147 "TsF" is not secreted actively and is clearly not antigen specific (141), as demonstrated by the way that purification of BW vs. AlO by binding to P815 Ag columns can separate the AlO from the BW effects (figures 45, 46 vs. figures 47, 48).  The observation of BW "TsF" molecules is of special  interest to investigators in the field of suppression due to the almost exclusive reliance on the BW5147 line for the fusion partner in the generation of suppressor hybrids.  15:7 Chapter Summary  In this study, we undertook to examine the effects of active immunization of DBA/2 mice with affinity purified AlO material, on the growth of tumors syngeneic to these mice.  We have shown that such  protocols do enhance tumor resistance and that this kind of treatment can result in both specific and non-specific anti-suppressor immune modulation.  Possible mechanisms for this phenomenon and the role of  anti-suppressor responses involved i n tumor growth are discussed.  158 Thesis Summary  In the past 30 years, there has been a notable lack of progress In • cancer therapy.  In 1956, the 5 year survival rate for the three major  k i l l e r s - colon-rectal, breast and lung cancers - was 44, 60 and 4 percent, respectively.  In 1986, these figures are 44, 64, and 5 percent.  While marked progress was obtained in this period for some rare cancers such as childhood leukaemia and Hodgkin's disease, these account for a tiny proportion of total cancer deaths.  In fact, deaths from the big  three outnumber easily deaths from a l l other cancers (all 200 + of them) combined. In terms of long-term survival, then, chemotherapy has clearly proved inadequate.  I.  Suppressor Therapy Two major observations have been made in the preceding chapters which  have potential significance regarding cancer therapy.  First,  tumor-specific immune suppression which blocks anti-tumor responses in the host animal can be demonstrated and second, the generation of this suppression and the associated soluble TsF molecule - can be interfered with in such a way that i t can impart increased tumor-resistance to the animal. As detailed in the f i r s t chapter, immunological approaches to cancer therapy have larged failed in the past.  While the precise regulatory role  of suppressors are s t i l l controversial, they have been implicated by  159  several investigators (148) as being involved in the maintenance of tolerance to self.  Since any attack by the body's immune system on a  tumor can be largely regarded as an exercise in autoimmunity (tumor cells are remarkable only for their indistinguishability from normal cells) (122) overcoming suppressors is an essential f i r s t step in initiating an anti-tumor immune response.  Thus, previous attempts at tumor therapy by  boosting the animal immunologically may have been misguided, to say the least.  Any increase in immune activity directed to the tumor would be  interpreted as autoimmunity ( i f this hypothesis is correct), and overwhelmed by a corresponding increase in the activity of the suppressor population.  This limitation applies to approaches designed to  non-specifically activate immunity (BCG, 11-1, etc.) and to approaches designed to specifically stimulate tumor immunity (vaccination with cancer cells or antigens). A system which appears at present (a very limited scope of clinical work has yet been done) to be independent of this system is the LAK-I1-2 approach.  Incubating immune cells with 11-2 in vitro appears to expand  this population to levels such that they can overwhelm suppression when infused back into the host.  The effect of 11-2 on suppressors i s unknown;  however, no TsC hybridoma has been demonstrated to produce 11-2 upon antigen stimulation, and TsC lines (dependent on 11-2) have yet to be conclusively demonstrated by any investigator.  If TsC populations are  independent of 11-2, that i s , require some alternate interleukin ("Il-S") for growth, then the results obtained with LAK therapy make sense; only Th and CTL's expand upon in vitro stimulation, and override nascent TsC populations when infused back into the patient.  160 Clearly then, what ever system of immunotherapy is to be attempted, i t is advantageous i f suppressors are dealt with f i r s t .  Two possible methods  for inhibiting the development of suppressors have been detailed in the preceding chapters. The f i r s t involves the use of monoclonal TsF administered in adjuvant in order to initiate an anti-suppressor response.  This procedure was  found to interfere with the generation of suppression to several unrelated tumors.  A potentially more powerful approach involves the purging of  suppressor populations using the B16G monoclonal coupled to toxins (as described in Chapter 4).  This approach has been demonstrated by other  investigators to have some validity using oI-J antiserum.  Unlike a l - J  sera, which can only be raised in a few strains of mice, and whose origin is s t i l l under debate, the B16G monoclonal is demonstrably active against both human and murine suppressor cells.  Thus, this monoclonal may be  useful for direct purging or to isolate polyclonal HTsF for use in TsF immunization therapy in humans. These approaches may be used in conjunction with the other immunopotentiating  processes in order to magnify the effect.  Of  particular value may be the simultaneous in vivo administration of B16G-Hp and 11-2.  Specific deletion of tumor reactive TsC populations may also be  accomplished using a-id Ab-toxin conjugates directed to the TsC cell surface receptors. This would avoid any possible deleterious effects that may result in self tolerance regulation from whole suppressor population purging.  161 II.  AlO and the T 3 C Receptor  In 1984, Davis and Mak simultaneously published the sequence of the T-cell receptor for antigen, which both groups had isolated by recombinant DNA techniques.  Subsequently, however, i t became clear that c e l l lines  which had been identified as suppressor hybrids lacked rearrangement of the appropriate segment of genome (38). There are two interpretations of these results; f i r s t , that suppressors do not exist; or second, that they have receptors coded for by genes removed from the region identified as coding for Th and CTL receptors. Extensive analysis of soluble suppressor factors has generally proven impossible to date, mainly due to a lack of available material for sequence analysis.  Without reagents (monoclonals) available which can  directly detect the presence of TsF molecules, investigators i n the field must laboriously detect TsF-producing hybridomas by analysis of their biological activity jLn vitro.  The availability of the B16G MAb has made  i t possible to screen thousands of T-cell hybrids by ELISA analysis in order to identify lines which produce constitutively high levels of the molecule.  The hybridoma AlO, because of i t s previously discussed  characteristics as a monoclonal suppressor with specificity for P815 TAA, is one of these hybrids. The yield in culture from one l i t r e of 'spent  1  medium is approximately 200 pg of isolable TsF.  Despite numerous  problems with analysis (the molecule is extremely labile and susceptible to protease lysis, as well as being glycosylated and N-terminal blocked) sequencing this molecule i s possible.  What resemblance this soluble TsF  162  molecule bears to the TsC antigen receptor is a question under debate; both bind nominal antigen.  Since B166 can bind to the surface of TsC, and  can be used to remove, by panning, suppressor c e l l populations (112) there is reason to suppose that similarities exist between the TsC receptor and the soluble TsF, in a situation analogous to secreted vs. membrane bound Ig.  Therefore, sequencing of the TsF molecule may lead directly to the  DNA coding for the T-suppressor c e l l antigen receptor, using well established recombinant DNA techniques. Additional T-cell hybrids producing TsF's directed to other antigens (which are well characterized, unlike P815 TAA) have been raised in our laboratory.  Again, thousands of prospective T-cell hybrids were screened  to select for constitutive over-producers of TsF.  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