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Invitro alteration of rat pancreatic islet immunogenicity in an allogeneic transplant model Skarsgard, Erik David 1991

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'TN VITRO A L T E R A T I O N OF R A T PANCREATIC ISLET IMMUNOGENICITY IN A N ALLOGENEIC TRANSPLANT M O D E L " by ERIK DAVID SKARSGARD M . D . , The University of British Columbia, 1985 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T O F T H E REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in T H E F A C U L T Y OF G R A D U A T E STUDIES (Department  We  of  Surgery)  accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH COLUMBIA May  1991  © Erik David Skarsgard  In  presenting  degree at the  this  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make it  agree that permission for extensive  scholarly purposes may be her  for  It  is  granted  by the  understood  that  head of copying  my or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department  of  SURGERY  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  June 3. 1991  ii ABSTRACT: Allograft rejection remains the fundamental stumbling block to tissue transplantation.  Traditional assumption has been that  transplanted tissue alone provides an antigen  source  (alloantigen),  which directly stimulates a host response resulting in graft rejection; accordingly, traditional attempts at circumventing the response have focussed immunosuppression.  on techniques  allograft  of recipient  Recently, increasing attention has been given to  a subset of non-parenchymal, bone marrow derived lymphoid cells (characterized by their surface expression of class II M H C antigen) which are carried passively with the allograft into an immune competent recipient.  A current hypothesis is that these cells, called  antigen presenting cells (APCs), participate in the sensitization immunologically naive but responsive leading ultimately to graft rejection.  of the  host to the transplanted tissue, Therefore, it has been  suggested that depletion of APCs from donor tissue prior to transplantation may permit allogeneic without host immunosuppression.  transplantation to occur,  In contrast to solid organs,  pancreatic islets are well suited to this type of immunomodulation prior to transplantation, since they can be maintained in a functional ex vivo state by cell culture. The purpose of this thesis was to evaluate donor islet A P C depletion  by pre-transplant cell culture and APC-ablative  photodynamic therapy (PDT), and to see whether either in vitro technique could prevent rejection in a rat,  allogeneic  transplant  model. Briefly, a donor (Sprague Dawley, R T l u ) -recipient (Wistar Furth, R T l a ) pair with a major histo-incompatible barrier was selected.  After collagenase digestion of donor pancreata, islets were  isolated from the digested tissue by centrifugation through a A  discontinuous  dextran gradient followed by hand picking using a  dissecting microscope.  Once isolated, the islets were either used  fresh, placed in tissue culture (Ham's F-12 media, 11 m M glucose, 5% C02/room air at 37 C) for variable periods, or subjected to A P C ablative P D T .  i ii  Islet A P C depletion was assessed by fluorescent immunocytochemistry.  Fresh, cultured and P D T treated islets were  frozen in liquid N2 then cryostat sectioned and stained for class II M H C + cells (APCs), using an anti-class II mouse monoclonal antibody (OX-6), followed by a fluorescent (fluorescein indothiocyanate) labelled anti-mouse monoclonal.  Using this technique, APCs could be  identified by fluorescent microscopy on the basis of their enhanced surface staining.  While fresh islets demonstrated between  1 and 5  APCs per cryostat section, a culture period of at least 10 days resulted in complete islet A P C elimination.  Islet allograft studies  with fresh and cultured islets were then performed to determine:  1)  if pre-transplant islet culture could sufficiently reduce donor tissue immunogenicity to allow successful allografting in immunecompetent recipients, and if so, 2) what duration of culture was necessary to permit consistently successful allografting.  Allografts of  fresh and cultured (4, 7, 10, 14, and 21 day) islets were placed under the renal capsule of immune-competent, recipient rats and after 12 days the grafts were removed and studied histologically for evidence of rejection.  While all grafts which were cultured for 10 days or less  prior to transplantation were rejected, 4/10-14 day cultured islets, and  4/5-21 day cultured islets demonstrated engraftment.  In vivo  function of 21 day cultured islet allografts was demonstrated by transplantation of islets via the portal vein, into recipients which had been rendered hyperglycemic by IV streptozotocin.  This resulted in  an immediate and sustained reversion to euglycemia (as assessed by daily plasma glucose determinations using a glucose analyser) over a 30 day period of study.  In contrast, streptozotocin "diabetic"  recipients of fresh and 14 day cultured islet allografts demonstrated a brief (7-10 day) period of graft function (euglycemia) prior to a return of hyperglycemia, consistent  with graft rejection.  Photodynamic therapy (PDT) achieves selective cell ablation by the stimulated emission of singlet oxygen from a light-activated compound (benzoporphyrin) which has been delivered to the cell target.  In these experiments, A P C elimination was attempted by in  vitro islet treatment with OX-6, followed by a specific, secondary antibody (RAMIg) to which B P D had been conjugated.  After U V light  iv  activation the treated islets were frozen, cryostat sectioned and immunostained for Class II M H C + cells.  In contrast to control islets  which underwent a secondary incubation with either B P D alone or BPD conjugated to an irrelevant secondary antibody, islets which underwent P D T using the specific RAMIg-BPD conjugate demonstrated elimination of APCs as assessed by immunocytochemistry.  When syngeneic  and allogeneic  transplants  were performed using islets which had undergone A P C "photoablation", the histologic appearance of the grafts was compatible with either inflammation in response to non-viable tissue, or allograft rejection. The temporal disparity between the duration of tissue culture necessary to deplete islet APCs and that required to allow islet allografting can be variably explained.  successful  One possibility is that  failure to stain APCs after a 7-10 day period of culture is not proof that these cells have been destroyed.  It is conceivable that culture  alters the surface of the A P C such that it is no longer identified by anti-Class II M H C immunostains, but nevertheless to present alloantigen.  retains its ability  Alternatively, one can hypothesize that in  vitro culture causes some donor tissue alteration other than A P C depletion which renders it less immunogenic. permit successful  syngeneic  or allogeneic  The failure of P D T to  transplantation despite  its  apparent ability to eliminate islet APCs suggests that the treatment itself may cause irreversible islet injury, and that the inflammatory reaction observed is merely in response to non-viable transplanted tissue.  V T A B L E O F CONTENTS:  page  ABSTRACT  Ji  ACKNOWLEDGEMENT I. 1.  2.  ix  INTRODUCTION: Transplantation Immunobiology:  Historical Aspects  1.1  Contribution from Studies of Tumor Immunity  1  1.2  Snell's Passenger Leukocyte Hypothesis  2  1.3  Passenger Cells and Graft vs Host Reactions  3  1.4  Lafferty's Contribution to the Allograft Reaction  4  1.5  The "Two Signal" Theory of T-cell Activation  5  The Major Histocompatibility Complex (MHC) and Alloantigen Presentation  3.  4.  5.  Allograft Rejection:  Current Concepts  3.1  The "Afferent Arc"  3.2  Allograft Rejection:  Immunologic  6  8 Effector Mechanisms  9  Tolerance  4.1  Passive and Active Tolerance  12  4.2  Induction of Allograft Tolerance in Adult Animals  13  Experimental Reduction of Allograft  Immunogenicity  5.1  Thyroid Transplantation Studies  5.2  Pancreatic Islet Transplantation:  14 Early Experience  with In Vitro Culture 5.3 5.4 5.5  Temperature as a Variable in Islet Culture In Vitro Use of Class II M H C Antisera Donor Tissue Irradiation  ;  II. E X P E R I M E N T A L R A T I O N A L E A N D PURPOSE  15 16 16 17 18  III. M A T E R I A L S A N D METHODS: 1.  Animals  20  2.  Islet Harvest Technique  20  3.  Islet Culture Technique  „2 0  4.  Islet A P C Depletion by Photodynamic Therapy (PDT), Using BPD-Antibody Conjugate 4.1  Antibody Conjugation Protocol  ',  21  4.2  Assessment of RAMIg Retention of BPD after Dialysis using 14c BPD  22  4.3  Islet Treatment with BPD-Antibody Conjugate  23  4.4  Photoactivation of BPD-bound Islets  24  5.  Evaluation of A P C Depletion by Immunocytochemistry  6.  Transplant Histology Studies,,  7.  Cultured Islet Allotransplantation of  25 „2 5  Streptozotocin-diabetic  Wistar Furth Recipients  26  IV. RESULTS: 1.  2.  Islet Immunomodulation by In Vitro Culture 1.1  A P C Depletion Experiments  ;  27  1.2  Islet Allograft Histology Studies  27  1.3  In Vivo Islet Allograft Function  28  Islet Immunomodulation by Photodynamic Therapy (PDT) 2.1 2.2  A P C Depletion Experiments Islet Transplant Experiments  V. DISCUSSION:  VI. BIBLIOGRAPHY:  29 29 30  _  35  LIST O F T A B L E S : Table 1: Post-dialysis RAMIg Retention of  1 4  C-BPD  Table 2:  Islet Treatment with Monoclonal Ab-bound B P D  Table 3:  Islet A P C Depletion and Allograft Histology Experiments  LIST O F FIGURES:  Figure la: Figure lb:  page  Fresh SD islets immunostained for Ia+ cells 7 day cultured SD islets immunostained for Ia+ cells  Figure 2:  41  ;  14 day cultured SD islets immunostained for Ia+ cells  Figure 3:  47  In vivo graft function in Streptozotocin diabetic W F recipients of 21 day cultured SD islets  Figure 9:  46  In vivo graft function in Streptozotocin diabetic W F recipients of 14 day cultured SD islets  Figure 8:  45  In vivo graft function in Streptozotocin diabetic W F recipient of fresh SD islets  Figure 7:  44  Successful cultured islet allograft immunoperoxidase-stained for insulin  Figure 6:  43  Successful cultured islet allograft at 12 days post-transplant  Figure 5:  42  Rejected fresh islet allograft at 12 days post-transplant  Figure 4:  41  48  SD islets immunostained for Ia+ cells following photodynamic therapy (PDT) with specific RAMIg-BPD conjugate  Figure 10:  49  SD islets immunostained for Ia+ cells following photodynamic therapy with irrelevant antibody (GA7sIg)-BPD conjugate  50  ix ACKNOWLEDGEMENT: I would like to thank my research supervisor, Dr. Mark Meloche for his patience and support during my year in the lab, and during the writing of this thesis.  In addition, I wish to thank Dr.  Andrew Seal, director of the M.Sc. program, for his ongoing guidance and  encouragement,  as well as Debra Kaminski for sharing her  technical and scientific lab expertise.  Finally, special thanks go to  Catriona Jamieson and Dr. Julia Levy for their assistance and guidance with the islet photoablation work, and to Quadralogic Technologies for providing the BPD.  page 1 INTRODUCTION 1) T R A N S P L A N T A T I O N IMMUNOBIOLOGY: HISTORICAL ASPECTS 1.1  Contribution from Studies of Tumor Immunity The  "immunity theory" of graft rejection was postulated by  several authors during the first decade of the twentieth based on histologic  century,  studies of rejected tumor homografts from  immunologically naive but competent recipients.  Popular belief at  the time was that all immune reactions were the work of circulating antibody, however the inability to demonstrate antibodies in hosts of allografts of normal tissue (versus tumor allografts), and failure to confer allograft immunity passively with this theory (1).  serum led to questioning of  A number of investigators, led by Murphy (2),  recognized the consistent presence of the "small lymphocyte" in host tissues surrounding rejecting transplanted tumors, and the concept of a cellular response in homograft rejection evolved. observed that immunity to tumor homografts  Mitchison (3,4),  could be transmitted  between identical mouse strains by the transfer of lymph nodes draining the site of the graft.  This resulted in a second set response  (an accelerated rejection phenomenon) received its first tumor homograft. this phenomenon  recipient  Billingham and colleagues termed  "adoptive transfer of immunity" (5).  Further experiments factor.  when the sensitized  sought  to characterize this  transferable  It was initially proposed that the lymph nodes were  transferring living tumor cells or at least tumor antigens (isoantigens).  The immunizing activity of transferred lymph nodes  seemed to be dependent  on the interval between administration of  the tumor implant and harvest of immunizing lymph nodes.  Activity  was maximum at 5-10 days, but had disappeared by 15-20 days.  It  was later shown that transfer of immunity could be accomplished not only by regionally draining lymph nodes, but also by spleen and remote lymph nodes as well (6).  Weaver et al investigated the  growth of transplantable tumors in diffusion chambers (permeable body fluids but not cells), placed intraabdominally, and found that tumor homografts were killed rapidly only if the diffusion chamber contained pieces of immune spleen (7).  These experiments, along  to  page  2  with the empiric histologic observation of lymphocyte penetration of homografts prior to destruction suggested an intimate interaction between immune lymphocytes and target tumor cells. In 1937,  a humoral homograft response was recognized when  Gorer reported that mouse sera from recipients of a rejected tumor homograft was capable of agglutinating red blood cells from the donor (8).  This also confirmed a belief that red cells and tumor cells  of the donor shared a common antigen.  However, when transfer of  tumor immunity to a secondary host with sera from a sensitized recipient was attempted, it was apparent that the growth of subsequent  tumor homograft was facilitated rather than inhibited,  compared to non-immunized controls (9,10).  Further experiments  revealed that lyophilized tumor and other non-living tissues (including spleen) from sensitized recipients were capable of enhancing tumor growth. It was speculated that perhaps the "enhancing effect" was confined to tumor homograft models on the basis of abnormal tumor growth potential, however the same effect was demonstrable with homografts of normal skin in both mouse and rabbit models (11,12). In most cases, homograft survival was prolonged relative to controls, yet there were only occasional reports of permanent graft survival (13).  Billingham (12), postulated that antisera either prevented or  more likely, delayed exposure of effective  homograft antigens to  regional lymph nodes, and referred to this phenomenon as "afferent inhibition" of the homograft reaction. 1.2  Snell's Passenger Leukocyte Hypothesis In a departure from traditional doctrine which stated that the  host response to tissue homograft was incited by  isoantigens  associated with the fixed, parenchymal cell population of the graft, Snell theorized that passively  transported, donor lymphoid cells were  responsible for the immunogenic stimulus that invoked the cellular homograft response.  He demonstrated that addition to the tumor  inoculum of normal lymphoid tissue of the same genotype as the tumor, would counteract the enhancing effect and result in tumor rejection (14).  He suggested that the added lymphocytes were able  page  3  to escape the afferent inhibition imposed by the presence of specific antisera, and reached regional lymph nodes where they could initiate a cellular immune response.  This concept was supported by the  known amoeboid motility of leukocytes  and the abundant lymphatic  supply of the skin, providing a mechanical basis for the rapid passage of lymphocytes from subcutaneous to regional lymph nodes.  or intracutaneous  grafts  Additional support for this hypothesis  came from Hardin and Werder, who noted that survival of skin homografts was prolonged by irradiation of the donor as well as the host, a treatment that would selectively  eliminate lymphoid cells  (15). 1.3  Passenger Cells and Graft versus Host Reactions The concept that donor lymphoid cells were capable of  mediating cellular immune responses was also supported by some early classic studies of graft versus host reactions (16,17,18).  It was  shown that a local response could be incited in guinea pig skin by intracutaneous injection of lymphocytes pre-sensitized to host tissue antigens  (the so-called Immune Lymphocyte Transfer [ILT] reaction),  and to a lesser extent by the innoculation of lymphocytes from unsensitized donors (Normal Lymphocyte Transfer [NLT] Reaction). Initially, there was some confusion as to whether the cellular immune response was donor or host in origin. Medawar assumed  Brent, Brown and  that the donor lymphocytes  cells in the host guinea pig's skin.  attacked  constitutive  Their finding that preirradiation of  guinea pig hosts with doses of up to 1500 rads did not prevent the development of N L T reactions, supported this hypothesis Subsequent work by other investigators models  (18).  using different animal  suggested that host lymphocytes were responsible for the  cellular response in both N L T and ILT reactions. experiments  An elegant series of  done by Ramseier and Billingham, demonstrated that  although non-lethal, total body irradiation of hamster hosts prior to intracutaneous injection of allogeneic,  sensitized  lymphocytes  markedly impaired the subsequent I L T reaction, this effect could be negated by the addition of an equal number of viable host lymphocytes to the normal or sensitized donor lymphocyte pool prior  page  to injection into irradiated hosts (19, 20).  4  Elkins innoculated the  renal subcapsular space of F - l hybrid rats with parental strain splenocytes and found that the resulting immune response could be prevented by total body irradiation or administration of leukopenic drugs such as cyclophosphamide and amethopterin to the host animal (21,  22).  Similarities were noted between local G V H reactions and the recently described in vitro Mixed Lymphocyte Interaction  System  (23), prompting Wilson and Elkins to suggest that in vivo mixed lymphocyte interactions were responsible G V H reactions 1.4  for the development  of  (24).  Lafferty's Contribution to the Allograft Reaction Conventional belief in the time of Thomas and Medawar, was  that transplanted tissue cells possessing allogeneic antigens were attacked by host lymphocytes mistakenly identified as tumor cells.  histocompatibility  because they were  Implicit to this concept of  immune surveillance was the notion that alloantigen alone directed the final differentiation of specific immunocyte clones.  According to  Medawar, the solution to allograft rejection involved immune manipulation of the host in attempts to dampen or eliminate completely  the host's response.  Lafferty (25), proposed that allogeneic responses are the result of a blood cellular interaction in which donor cells of the lymphocyte/macrophage lineage provide a stimulus for activation of specific receptor-bearing host immunocytes. stimulator-responder cell interactions  He hypothesized  that  result in activation and  proliferation of an effector cell clone if a histoincompatibility exists between the stimulator and responder cells.  Respecting the premise  that self stimulation of cellular immune responses is forbidden, Lafferty  suggests two models  incompatibility.  of stimulator-responder cell  In the first instance, donor stimulator cells which  accompany the graft are incompatible with the host responder cells, thus permitting responder cell activation.  In the second instance, if  both stimulator and responder cells are host in origin, modification of  page  5  the stimulator cell "self" antigen by some foreign agent (such as alloantigen),  is  necessary.  These two basic concepts can be expressed algebraically: (1)  S  + HRA  B  >  H R - A (initiation of immune response)  §A (2)  S  A  S  A  HRA  +  + ag a  g  S ag + A  >  negative  >  S  is not equal to H  R  >  A  H  a  g  S  A  R '  (resting  situation)  and if  A  then A  (initiation of immune response)  where S = stimulator cell of phenotype B (donor) or A (host), HRA  =  host responder cell, ^ R ' A  =  activated host responder cell, ag =  foreign tissue (alio) antigen, S a g = altered host stimulator cell. A  1.5  The "Two Signal" Theory of T-cell Activation Central to Lafferterian theory is a two signal model of T-cell  activation.  Foreign tissue antigen (alloantigen)  is processed and  presented on the surface of a stimulator or "antigen presenting" cell (APC), and is engaged by the responder T-cell receptor. constitutes "signal 1".  This  Also present on the surface of the A P C are  regulatory M H C antigens.  Lafferty believes that it is the engagement  of A P C "non-self" M H C antigens by the responder T-cell, which triggers "signal 2", also known as "costimulator activity":  the release  of interleukin-1 by the A P C . Thus, only after ligand binding (signal 1), has occurred in conjunction with release of an inductive molecule by the A P C (signal 2), does responder T-cell activation with specific clonal effector cell expansion occur.  It is assumed that this antigenic  property of M H C antigen is distinct from that of alloantigen, as it has been suggested that M H C antigens by themselves, may only be weakly  immunogenic  (26).  It is now conceptually possible to propose mechanisms of responder or "host" T-cell activation by either donor (allogeneic) or host (syngeneic) APCs.  If the APCs are of donor origin, M H C  page 6 incompatibility will facilitate costimulator activity by the A P C result in the initiation of an allograft response.  and  This premise  provides rationale for experimental depletion of APCs  from donor  tissue prior to transplantation, in an attempt to circumvent allograft rejection.  Alternatively, if the APCs are host in origin, some  modification of surface M H C antigen (perhaps through binding with alloantigen),  is necessary to produce the requisite  stimulator/responder occur.  incompatibility for the  allograft  response to  This mechanism has become known as the "Alternate  Pathway  of Alloantigen  Presentation."  2) T H E MAJOR HISTOCOMPATIBILITY C O M P L E X (MHC) A N D A L L O A N T I G E N PRESENTATION The major histocompatibility complex (MHC), is that part of an organism's genome which encodes for the production of cell surface proteins called M H C antigens, which are believed to play a regulatory role in cell mediated immune responses.  The M H C has been best  characterized in the mouse, where there appear to be distinct histocompatibility loci located on virtually every chromosome.  One  of these loci designated H-2, exerts a particularly strong effect on allograft rejection, and is called a major histocompatibility  locus  while the others are referred to as minor histocompatibility loci.  An  important characteristic of the H-2 locus is the enormous genetic polymorphism that exists due to allelic diversity in outbred species, as well as the not infrequent chromosomal recombination that occurs during  meiosis.  The  availability of inbred mouse strains, alloantisera and  monoclonal antibodies has permitted mapping of the M H C genes. M H C is divided into two major subclasses: H-2 complex and (b) the T l a complex. four regions: regions:  K,I,S and D,  The  (a) the classically defined  The H-2 complex contains  while the T l a complex contains three  Qa-2,3, T l a and Qa-1 (27, 28).  M H C antigens are divided into two classes:  Class I and Class II.  The class I molecules include the transplantation antigens K , D and L , and  consist of a transmembrane glycoprotein which is  linked to a 6-2 microglobulin on the cell surface.  noncovalently  These molecules  page  have five distinct regions: membrane, one domain.  7  three globular domains above the cell  transmembrane domain and one  Carbohydrate residues  intra-cytoplasmic  are attached to the external domains.  Class I M H C antigens are found on virtually every nucleated somatic cell and provide the essential context of self in which foreign cell surface antigens (such as those produced by a viral infection), can be recognized and destroyed by cytotoxic T-cells (designated CD8+ cells). This phenomenon, which results in direct activation and proliferation of a clone of effector cytotoxic T-cells is termed class I M H C restricted  alloantigen  presentation.  The class II genes Aa, AB, Ea and EB are located in the I region (29).  Class II molecules (also known as la for I region "associated" ),  consist of two polypeptide chains (a,8) held together by non-covalent interactions.  These molecules  are also transmembrane globular  glycoproteins  with two external domains, one transmembrane  domain and one intra-cytoplasmic domain.  Class II antigens are  found primarily on bone marrow derived lymphoreticular cells (activated B and T cells, macrophages and dendritic cells), as well as on vascular endothelium.  These molecules  provide  self-recognition  elements that allow macrophages and dendritic cells ("antigen presenting" or "accessory" cells) to interact, in the presence of processed, foreign antigen, with responder T-cells.  The result is the  generation of activated T-cells of the helper or CD4+ subset, which subsequently  participate in the production of either antibody-  secreting plasma cells or cytotoxic T-cells. presentation  This type of alloantigen  is termed class II MHC-restricted  presentation.  These M H C molecules show structural homology with the immunoglobulin receptor of the B-cell, the T-cell receptor and the thy-1 molecule  (T-cell differentiation antigen),  surface of mouse T cells.  expressed  on the  This homology suggests that the genes  encoding these different molecules  share a common ancestor, and  that marked changes have occurred after divergence of the genes to fulfill different functions.  These genes are referred to collectively as  "the Super gene Family." The M H C s of mice and other species differ fundamentally only in the organization of their genes and the descriptive nomenclature  page  (30).  8  In rats, the M H C consists of four major class I loci and two class  II loci, and carries an RT1 designate (31).  The M H C of humans is  referred to as the H L A complex and is located on the short arm of chromosome 6 (32).  Human class I transplantation antigens are  designated A , B, and C and the class II antigens are designated DP, D Q and DR. 3) A L L O G R A F T REJECTION: CURRENT CONCEPTS 3.1  The "Afferent Arc" Host recognition of immunogenic determinants on allografted  tissue initiates an immune response.  The first phase of this response,  called the "Afferent Arc", begins with an encounter between graft alloantigen (which may be present in blood, lymphoid tissue, or within the graft itself), and the appropriate host helper T-cell.  Donor  antigen presenting cells (APCs) called dendritic cells, carried passively with the graft are capable of self-processing and  alloantigen  can efficiently present antigen to host lymphoid cells.  (33).  Alternatively, alloantigen can be processed and presented by host APCs (34, 35), as is the usual case for antigen present on parenchymal cells of transplanted tissue.  The mechanism by which  an A P C presents antigen to a responsive lymphocyte is unclear.  Most  evidence suggests that antigen is somehow modified by the A P C prior to presentation (36).  As suggested by Lafferty, if the A P C is  host in origin, antigen processing must impose an alteration in surface M H C antigens to facilitate a stimulator / responder incompatibility, which results in responder-cell activation. The route of host sensitization depends on whether or not the graft is vascularized.  With vascularized grafts, host cells within the  blood compartment will be the first to encounter graft antigens, whereas in the case of skin grafts, cells within draining lymph nodes are first to contact antigen. The most striking aspect of the immune response is its specificity.  For each immune stimulus, a distinct population of  antibodies or immune cells are elicited, which suggests that there must be specific antigen receptors (with structural similarity to immunoglobulin) on the surface of naive responder T-cells.  How  page 9 these recognition molecules develop is unknown.  The clonal selection  theory suggests that clones of lymphocytes specific to an antigen probably arise by somatic mutation prior to antigen encounter. subsequent  encounter between antigen and specific,  stimulates  proliferation and maturation of that clone.  dedicated  The clone  Clonal response to alloantigen can be either B or T cell in origin. Sensitized B cells proliferate and differentiate into plasma cells that actively secrete antibody, while sensitized T cells proliferate into a clone of T cells capable of inflicting damage to the graft by virtue of their close range.  The activation of resting small lymphocytes of  both types occurs in regional lymph nodes and the spleen. 3.2  Allograft Rejection: Early investigators  Effector Mechanisms studying the mechanism of graft rejection  examined histologic sections, and the presence of a specific type of cell was taken as evidence of its role in allograft rejection (37). recent availibilty of monoclonal antibodies against specific  The  T-cell  subsets has facilitated identification of the relative proportion of cytotoxic/suppressor (CD8+) cells to helper/inducer (CD4+) cells in stable grafts, as well as those undergoing rejection. The different types of rejection reactions have been best characterized in the kidney.  Classic, acute rejection involves a  lymphocytic infiltration of the renal interstitium and blood vessels. Small lymphocytes are seen in contact with peritubular capillary and venular endothelial cells within hours of transplantation. three days, large lymphocytes  appear adjacent to the endothelial  cells lining intertubular capillaries and venules, injury becomes evident.  Within  and endothelial  The lymphocytes infiltrate  throughout the interstitium, with progressive  diffusely  disruption of  peritubular capillaries and venules and interstitial fluid accumulation, leading to a fall in renal blood flow and further cellular damage on an ischemic basis. Hyperacute rejection is antibody mediated and is seen in recipients with antidonor antibodies  at the time of transplantation.  This is most likely to occur in humans when exposure to blood transfusions, pregnancy, or a previous transplant has induced the  page formation of antibody to class I antigens.  10  Preformed A B O antibodies  can also result in hyperacute rejection of most incompatible organs, whereas antibodies to class II antigens do not (38). antibody binds specific resulting complement (39).  Typically,  antigen on the vascular endothelium, with activation and massive  intravascular activation  Biopsies of hyperacutely rejected kidneys show deposits of IgG  and C3 on the glomerular and peritubular capillary walls, with luminal occlusion  by platelet-fibrin aggregates.  Hyperacute rejection can also occur following transplantation between phylogenetically distant species, such as a kidney graft from a pig to a dog (40).  Apparently the recipient has natural antibodies  against the donor species without previous antigenic  exposure.  Chronic low-grade rejection occurs in most allograft recipients and results in gradual loss of organ function over months or years. Histologically, this involves interstitial fibrosis and chronic vascular changes with arteriolar narrowing and thickening of capillary basement  membranes caused by deposition of antibody and  complement,  with secondary  fibrosis.  Cytolytic T-cells have always been considered to be the primary effectors in allograft rejection because of their demonstrable activity in vitro (41, 42).  Anti-donor specific cytolytic cells have  been retrieved from human renal, hepatic and cardiac allografts, as well as many animal transplant models (43, 44, 45). Monoclonal antibodies directed against mouse T-cell subsets have been used to determine the relative contributions of these subsets to graft rejection.  Cobold and Waldmann have found that  when anti-L3T4 monoclonal antibody (directed against  surface  markers on helper cells), was administered early to skin grafted mice, significant graft prolongation was achieved, while anti-Lyt 2 antibody (directed against markers on cytolytic T-cells), effect.  had no  Both anti-L3T4 and anti-Lyt 2 significantly prolonged graft  survival when administered later (46).  These experiments  support  the central role of the T-helper cell early in the allograft response, and suggest that the cytolytic T-cell is more important later on. Recent attention has been given to the role of Interleukin 2 (II2) receptor- bearing cells in allograft rejection.  Administration of  page  11  monoclonal antibody against mouse 11-2 receptor significantly prolonged vascularized cardiac allograft survival in two separate H-2 incompatible strains (47),  and similar experiments  results have been reported in the rat (48).  with comparable  The 11-2 receptor is  expressed on all activated T-cells, and the production of 11-2 (which is a lymphokine mediator of T-cell activity) by activated T helper cells (49), suggests a central role for the T helper cell as the initiator of  cytolysis. Cell killing in allograft rejection can be accomplished by a  specific cytolytic effector clone or none-specifically, through the release of a variety of inflammatory mediators by activated helper cells, resulting in a Delayed-Type Hypersensitivity (DTH), reaction. Support for the latter is provided by studies which have shown that T-cell deprived rats can reject skin, heart or renal allografts when reconstituted solely with helper T-cells (50, 51).  The identification of  rat lymphotoxin in rejecting rat renal allografts has led to its incrimination as the actual injurious agent in D T H reactions  (52),  while others have suggested that the cytotoxic effect of lymphotoxin is augmented in the presence stimulated helper cells (53).  of gamma-interferon secreted by Since gamma-interferon also induces  expression of class II antigens on parenchymal cells, lymphotoxin and  gamma-interferon may have synergistic deleterious  transplanted  effects on  tissue.  Tests of in vitro cytotoxicity against various tumor targets has shown that lymphoid cells from non-sensitized animals can be highly cytotoxic to certain targets (54) .  This activity shows no evidence of  target cell specificity or memory, and has been attributed to the activity of N K cells.  N K cells are nonadherent, nonphagocytic and do  not express surface immunoglobulin; other cells under M H C restriction.  nor is their interaction with  Any stimulation of an animal's  immune system seems to result in an increase in N K activity, likely through lymphokine (gamma interferon) release.  Since N K cells will  only lyse a limited range of target cells, their in vivo significance remains  unclear.  Antibody-mediated  allograft  damage,  apart from  hyperacute  rejection is of uncertain significance, although it has been identified  page in a number of models (55, 56). involves and  The effect exerted by the antibodies  a number of non-specific  antibody-dependent chemotactic  cytotoxicity,  12  pathways, including complement,  clotting and generation  of kinins  factors.  4) IMMUNOLOGIC T O L E R A N C E 4.1  Passive and Active Tolerance Tolerance is any specifically altered state of reactivity that  results in the failure of the animal to express an immune response to the tolerizing antigen, while leaving responses to unrelated antigens intact.  Burnet's clonal selection theory postulated that tolerance to  "self" antigens occurs during the development of the immune system, as a result of deletion of self-reactive clones (57). supported by neonatally  This theory is  induced, (passive) transplantation  tolerance,  in which induction of tolerance in strain A mice results from neonatal injection of (A x B) F l bone marrow.  Adult mice treated in this way  will accept skin grafts from strain B mice, but reject skin grafts from third party animals in a normal manner.  Similarly lymphoid cells  from such tolerized animals will not respond to strain B cells in a mixed lymphocyte reaction, yet respond normally to third party cells.  A n identical form of tolerance occurs when adult animals are  lethally irradiated, and then reconstituted with F l bone marrow; called "Radiation Chimeras."  so-  Tolerance of this form cannot be  transferred from one animal to another, and is most likely due to deletion of a clone of responder T-cells; Tolerance,"  a form of "Passive  (58).  A second form of tolerance can be induced by exposure of the immune system to soluble antigen, either during neonatal life or, in some cases following appropriate antigen administration to the adult animal.  Neonatal tolerance is maintained as long as the tolerizing  antigen, which is mistakenly identified as self, is present.  Tolerance  to that antigen can be reversed by its withdrawal, suggesting a clonal deletion mechanism.  Tolerance in adult animals can be induced by  injection of very low or very high doses of soluble antigen.  This  mechanism of tolerance induction is referred to as "active" tolerance  page  13  and involves T-suppressor cells, and can be transferred to a naive animal by T-lymphocytes from a tolerant donor (59). The exact mechanism of T-suppressor cell induction and function is unclear.  It has been postulated that induction involves an  MHC-restricted cellular interaction between a naive suppressor cell and an antigen-activated accessory cell (APC), or T-helper cell.  The  result is an activated "effector" T-suppressor cell which acts directly, or via secreted immune  suppressor proteins to suppress  responsiveness  antigen-specific  (60).  It should be recalled that there are other ways of suppressing the immune response, one being the presence of antigen specific antibody, which removes  antigen and thereby diminishes host  reactivity, in both specific and non-specific (DTH) immune responses. 4.2  Induction of Allograft Tolerance in Adult Animals A successful allograft of tissue which has undergone  pretransplant modification to remove donor accessory cells, will undergo prompt rejection when the recipient is actively immunized with donor accessory cells.  However with time, the graft enters into  a stable interaction with the host and can no longer be rejected by active immunization of the recipient.  This stable  graft-host  interaction results from the induction of a state of specific altered immune reactivity ("allograft tolerance"), that allows acceptance of a graft that would otherwise be rejected. The mechanism of this tolerant state is unknown, but could involve either passive (clonal deletion), or active mechanisms alone, or in combination (61). spontaneous  (suppression)  The kinetics of  graft stabilization vary considerably according to the  tissue studied;  thyroid allografts stabilize more slowly than islet  allografts (350 days and 120 days, respectively),  (61).  Graft stabilization could conceivably occur in one of two ways. Either there is some adaptation of the graft such as loss of antigenicity, or the reactivity of the host is altered. hypothesis  is  The latter  supported by the observation that retransplanted  (cultured) thyroid allografts from spontaneously  stable animals into  naive recipients, are promptly rejected upon host immunization with  page "original" donor accessory cells. established,  This illustrates that the long-  cultured allograft still demonstrates  non-adapted  14  antigenicity  in a  host.  This form of specific tolerance has been demonstrated in animals carrying both stable islet and thyroid allografts (61).  These  animals accept a second, uncultured graft of donor type, but reject a third-party graft transplanted at the same time.  The acceptance of a  graft that would otherwise be rejected and the specificity of the graft acceptance reflect the specific  state of tolerance that has been  induced in the recipient of a cultured graft.  Although tolerant  animals are hyporesponsive in vivo they retain normal mixed leukocyte reactivity in vitro, suggesting that a clonal deletion mechanism is not responsible for tolerance induction under these circumstances (61).  Some active mechanism must be inhibiting in  vivo graft rejection. 5) EXPERIMENTAL REDUCTION OF GRAFT IMMUNOGENICITY 5.1  Thyroid Transplantation Studies The concept of organ graft pretreatment in an effort to  modulate its immunogenicity and prevent rejection has found its major experimental success in endocrine transplantation, due largely to the fact that endocrine grafts do not require immediate vascularization for continued survival. The  initial reports of prolonged allograft survival after a period  of pre transplant culture came from Lafferty, who maintained Balb/c mouse thyroid lobes in 95% 02, culture for variable periods, and then transplanted them under the renal capsule of H-2 incompatible, nonimmunosuppressed  C57B1  recipient mice (62, 63).  Graft function  was followed by measuring the level of 125i uptake by directed scintillation counting over the graft. clearly showed  These initial experiments  that thyroid tissue maintained in organ culture prior  to transplantation survived far longer in an allogeneic host than did non-cultured tissue.  Additional experiments  by Lafferty showed  that brief pretreatment of the host with cyclophosphamide prior to thyroid allografting allowed a significant reduction in the period of  page  15  organ culture (from four weeks to one), required to effect prolonged allograft  survival  (25).  Lafferty concluded that culture conditions of high oxygen concentration were selectively toxic to the vascular bed and to lymphoreticular elements of the graft, and proposed that organ culture may have removed from the graft those cells capable of providing an allogeneic complemented  stimulus (64).  Sollinger and associates  the findings of Lafferty with the discovery that  addition of high oxygen tension to the conditions of culture resulted in markedly prolonged thyroid xenograft immunosuppression  (65).  Further support for the concept by culture depletion  survival without host  of alloengraftment  facilitated  of donor lymphoreticular elements was  provided by Talmadge and colleagues (66),  who demonstrated  that  injection of only 1000 donor-type peritoneal cells into a mouse host carrying a cultured thyroid graft restored its immunogenicity, and led to prompt allograft rejection. 5.2  Pancreatic Islet Transplantation:  Early Experience with In Vitro  Culture After success with prolonged mouse and rat parathyroid allograft  survival after pretransplant culture under conditions  high oxygen tension was reported (25,  67), many  turned their attention to the possibility  of transplantation of  of  investigators  pancreatic islets with a view towards eventual application to the treatment  of diabetes  mellitus.  Kedinger et al, reported prolonged recipient survival, with biochemical evidence of graft function after transplantation of 4 day cultured rat islets directly into the liver of recipients rendered glucose intolerant by treatment with IV streptozotocin (68).  One of  the early problems with culture of islet tissue was the apparent sensitivity  of the parenchymal tissue to conditions of culture (most  notably high oxygen concentrations), function.  and the loss of endocrine  To reduce islet oxygen toxicity, Bowen and colleagues (69),  cultured mouse pancreatic islets in clusters of approximately 50 islets (so-called  "megaislets"), and reported prolonged, functional  page  allograft survival.  16  Lacy's group (70), introduced a technique of  collagenase digestion  of rat pancreas, with hand-picking of individual  islets which were maintained in 7 day, high oxygen culture prior to transplantation as xenografts  into mice.  The addition of a single dose  of anti-rat lymphocyte serum to the hosts prior to transplantation resulted in a significant prolongation of xenograft survival. 5.3  Temperature as a Variable in Pancreatic Islet Culture The discovery that lymphocytes  which were cultured at low  temperature (22C), lost their ability to stimulate in an M L R , but retained their ability to respond to non-cultured lymphocytes,  suggested that low  temperature  allogeneic  culture  adversely  affects the immunogenicity of lymphoreticular cells (71).  With this  information, Lacy and his colleagues (72), performed allogeneic rat islet transplants following 7 day culture at 24C in room air, and demonstrated 85% graft survival beyond 100 days.  Thus, it  appeared that low temperature had the same deleterious  effect on  the lymphoreticular elements in the islet cultures, as high oxygen concentration, but spared the parenchymal cells.  Lacy also proved  that the endocrine cells retained their antigenicity, by inducing acute rejection of functioning, tolerated allografts by the injection of donor peritoneal exudate cells 5.4  (73).  In Vitro Use of Class II M H C Antisera With the advent of monoclonal technology,  attempts were  made to eliminate immunogenic lymphoreticular cells from organ allografts with sera directed against class II M H C molecules. Faustman and colleagues demonstrated that class II M H C molecules were not expressed on the surface of mouse pancreatic B cells, but rather were on the passenger leukocytes present within the donor tissue, termed "dendritic" cells (74, 75). anti-class  Faustman then showed that  II (anti-dendritic cell) antibody and complement  treatment  of donor mouse pancreatic islets resulted in 100% survival of mouse islet allografts  for more than 200 days, following  across a major histocompatibility barrier(76). attempted  transplantation  Gores and colleagues  to reproduce these findings, but could not, and instead  page  17  described an elegant in vitro, mixed islet-lymphocyte coculture model which demonstrated that in addition to donor cells, recipient cells and even third party antigen presenting cells were capable of alloantigen presentation.  (77)  Another application of class II antisera to the elimination of la bearing antigen presenting cells has been the recent development of "immunotoxins", which are highly toxic proteins, such as ricin or diptheria toxin, that have been covalently coupled to monoclonal antibodies.  Shizuru et al (78), have shown that pretreatment of islets  with an anti-la monoclonal antibody covalently conjugated to purified ricin toxin, results in the elimination of the allostimulatory properties of islets in mixed lymphocyte islet cell cultures (as assessed by proliferative indices of responder lymphocytes),  without  damage to the hormone secreting cells. 5.5  Donor Tissue Irradiation The rationale for U V irradiation of donor tissue was the  recognition of the importance of passenger leukocytes  (specifically  dendritic cells), in the allograft response, and evidence that these cells were exquisitely sensitive to U V light inactivation. colleagues  Hardy and  irradiated isolated Lewis rat islets, "rested" them in tissue  culture for 24 hrs and then transplanted them into glucose intolerant, immunocompetent A C I hosts.  With this treatment regime,  he found that irradiated islet allografts corrected hyperglycemia for longer than one year in over 70% of recipients. subsequently  Furthermore, he was  able to induce rejection by the administration of  donor-type rat dendritic cells  (79).  page  18  RATIONALE: As outlined above, there is already a substantial body of experimental evidence which suggests that the donor antigen presenting cell (APC) plays an important role in the initiation and regulation of allograft rejection.  The recent availability of inbred  small animal strains and monoclonal antibodies  has permitted  mapping of the major histocompatibility complex (MHC), and identification of its gene products -called M H C antigens, on cell surfaces.  APCs are composed of a number of bone marrow derived  lymphoreticular cells (activated B and T cells, macrophages and dendritic cells), which are characterized by their unique of class II M H C (also known as la) antigen; cell surface enhancement treatment  synonomous  consequently, selective  seen on flourescent  with fluorescein-labelled  expression  microscopy  anti-la antibody is  with the presence of antigen presenting  after  now  cells.  As a caveat, one might infer that the inability to demonstrate selective cellular fluorescence  with anti-la  immunofluorescent  techniques is evidence of the absence of APCs within the tissue specimen.  This rationale allows objective evaluation of donor tissue  immunomodulation in which the aim of the treatment is depletion or complete elimination of APCs from the donor tissue. In Vitro Donor Islet Culture: The immunomodulatory effects of in vitro islet culture may be assessed in two ways.  First, culture depletion of isolated rat islet  APCs can be evaluated by fluorescent  anti-la islet staining after  increasing periods of in vitro culture.  In this manner, one might  determine the "critical period" of culture required to "remove" all islet APCs. allogeneic  Next, fresh and cultured islets are transplanted into hosts, and the grafts are subsequently  evidence of engraftment shown to engraft  versus rejection.  preferentially  assessed by histologic  evaluated for  If cultured islets are  over non-cultured controls  criteria), one might infer that pre-transplant,  in vitro culture imposes changes in the donor tissue that allotransplantation. successful  (as facilitates  If the duration of in vitro culture necessary for  allotransplantation is comparable to that necessary for  page  19  islet A P C depletion, this would provide strong supportive evidence for the role of the "donor" A P C as the sole mediator of the allograft rejection  response.  Elimination of Donor Islet APCs by Photodynamic Therapy (PDT): Benzoporphyrin derivative monoacid ring A (BPD-MA), is a tetrapyrrole ring with photosensitizing  properties.  When the  molecule is stimulated by light in the ultraviolet range (600nm), singlet oxygen is released causing destabilization and lipid peroxidation of nearby cell membranes, resulting in cell lysis and death.  If the BPD molecule could be conjugated to an anti-la  monoclonal antibody, then theoretically, isolated islets can be "purged" of donor APCs by incubation with the BPD-anti-Ia conjugate, followed by photoactivation of BPD with ultraviolet light. If the allograft response were mediated solely by donor APCs, this in vitro technique could be performed on isolated islets and may then allow  successful  islet allotransplantation.  PURPOSE:  The purpose of this thesis is to evaluate two techniques of in vitro immunomodulation of rat pancreatic islets in an transplant model:  1)  donor islet culture and 2)  ablation by Photodynamic therapy (PDT).  allogeneic  donor islet A P C  Finally, based on the  results, an assessment of the role of the donor A P C in the allograft response will be made.  page  20  MATERIALS AND METHODS: 1)  ANIMALS: Two  inbred strains of rats differing at both major and minor  loci of the major histocompatibility complex were used.  Sprague  Dawley (SD) rats (RTlu), were utilized as pancreatic islet donors, and Wistar Furth (WF) rats (RTla), as transplant recipients. 2) ISLET H A R V E S T TECHNIQUE: Male SD rats (200-250 gm), were anaesthetized with intraperitoneal urethane (100  mg/kg), and through a midline  laparotomy, cardiorespiratory arrest was pneumothoraces.  induced with bilateral  The proximal common bile duct was cannulated  with a fine polyethylene catheter and the duct was occluded distally, at the ampulla of Vater.  The pancreas was then distended in a  retrograde fashion with cold (4 C) collagenase (Type XI, Sigma Chemicals) in sterile Hanks' balanced salt solution (HBSS), at a concentration of 0.42 mg (650 U) per ml.  After in situ collagenase  distension, a total pancreatectomy was performed.  The glands were  digested for 22 minutes in a 37 C waterbath, and then the digestion process was terminated by the addition of sterile, cold HBSS. digested  The  glands were dispersed by trituration through a sterile,  siliconized pipette.  The crude tissue slurry was passed through a  200 Jim screen filter to remove undigested ducts, blood vessels and lymph nodes, and was then centrifuged through a discontinuous dextran (Sigma Chemicals) gradient consisting of two monolayers of specific gravity 1.065  and 1.031 respectively.  The less dense islet  tissue was then aspirated from the monolayer interface, washed with sterile HBSS, and then further purified by hand picking under a dissecting microscope.  Using this technique, 200-400  morphologically intact islets were isolated per pancreas. 3) ISLET C U L T U R E TECHNIQUE: After isolation, islets were either used immediately or subjected to in vitro culture (prior to subsequent use), for 4-21  days  in Ham's F-12 medium (Gibco, [glucose] = l l m M ) , supplemented with  page  21  25% fetal calf serum, 15 m M Hepes buffer and l%pen/strep/ fungizone, in a 5% C02/room air incubator at 37 C . The islet suspensions were agitated daily with a Pasteur pipette to prevent islet clumping, and the media was changed weekly for those islets cultured longer than 10 days. 4) ISLET APC DEPLETION B Y PHOTODYNAMIC T H E R A P Y (PDT), USING A BPD-MA -ANTIBODY CONJUGATE: 4.1  Antibody Conjugation Protocol: Benzoporphyrin derivative monoacid ring A ( B P D - M A , to be  subsequently  abbreviated as BPD), was produced by Quadra Logic  Technologies, Vancouver, B . C , and was stored frozen as a stock solution in dimethylsulfoxide (DMSO), at 1 mg/ml.  Immediately  prior to conjugation, the stock solution was diluted to 200 Mg/ml in sterile phosphate-buffered  saline (PBS), then was mixed with a  known quantity of monoclonal antibody.  For pilot experiments, BPD  was conjugated directly to mouse anti-rat la (OX-6), subsequent  experiments  demonstrated  however  better photoablation  of  APCs  when B P D was conjugated to a secondary antibody (Rat anti-mouse Ig = RAMIg ). The affinity between BPD and antibody is a non-covalent association between hydrophobic moieties of the 2 molecules, and relies on an aqueous milieu to maintain "binding."  A l l conjugations  were carried out in low light conditions to avoid photoactivation of BPD. Two specific RAMIg-BPD conjugates were prepared:  one with a  calculated BPD : Ab, molecular ratio of 15:1, and the other with a calculated ratio of 40:1.  (Molecular weights:  antibody *=* 150,000 g/mole).  B P D - M A = 718 g/mole,  These molecular ratios were selected  on the basis of previously performed photoablative  experiments  using a chronic granulocytic leukemia (CGL) cell line, in the laboratory of Dr J Levy (Dept of Microbiology, U B C ) . In addition, conjugates of BPD to an "irrelevant" monoclonal antibody (goat anti7s ribosomal protein Ig = GA7sIg), were prepared at the same two relative molecular ratios to serve as controls.  The antibody-BPD  page conjugates  22  were allowed to incubate for 1 hr at room temperature  after mixing, and were then dialyzed for 36 hours in eppendorf tubes (covered with a dialysis membrane impermeable to molecules  of  molecular weight > 14,000 g/mole), against 3 litres of PBS with a stir bar, at 4C.  In this manner, any unbound BPD would be dialysed  away on the basis its size relative to the dialysis membrane pores. 4.2  Assessment of RAMIg Retention of BPD after Dialysis using 14 C  labelled BPD: Liquid scintillation counting of ^^C-BPD  in a known quantity of  dialysed conjugate was carried out to determine the actual antibody : BPD molecular ratio in the conjugate following the 36 hour dialysis period.  RAMIg-BPD conjugation was carried out as described above  using 1 ^C-iabelled BPD with a known specific activity of 134 disintegrations per minute (dpm) per nanogram (ng). 0.1 ml samples of each conjugate (with calculated  After dialysis, molecular ratios  of 15:1 and 40:1, respectively), were mixed with 5 ml of aquasol (scintillation liquid), and each sample was counted in triplicate over a 5 minute period using a scintillation counter (Phillips Instruments). From the total number of counts, the amount of 14C-labelled B P D (in ng), in the aliquot of dialysed conjugate could be calculated using the formula: total # of counts (dpm)  =  amount of 14C-BPD-MA (ng)  activity of B P D - M A (134 dpm/ng) Knowledge of the quantity of RAMIg in the conjugate aliquot thus allowed calculation of the actual molecular ratio of BPD to antibody after dialysis.  The calculated (pre-dialysis) and actual  (post-dialysis)  molecular ratios of the two RAMIg-BPD conjugates, are shown in Table 1.  page  23  Table 1: Pre-dialysis Molecular  Post-dialysis  Ratio (RAMIg : BPD)  4.3  Ratio (  1 4  %BPD  Molecular  Retained  C-BPD)  1 : 40  1 : 18  45%  1 : 15  1 : 6.5  43%  Islet Treatment with BPD-antibody conjugate: Sprague Dawley islets were isolated in the usual fashion and  subjected to overnight culture in complete Ham's F12 medium at 37C in a 5% C 0 2 , room air incubator.  The following morning the islets  were resuspended and deposited in 96 well Costar plates  (200  jil/well), at a density of approximately 50-75 islets per well.  Using a  micropipette with the assistance of the dissecting microscope, the islets were washed under direct vision by 2 complete  volume  exchanges with sterile PBS, to remove extraneous protein.  The islets  were then resuspended in the 200 j i l wells with l l m M Ham's F-12 + 1% pen/strep/fungizone,  (no fetal calf serum), and incubation with  primary antibody was carried out. The primary incubation was performed using purified O X - 6 Ig obtained from an ammonium sulfate-cut, McMaster;  ascites preparation (Dr R  Dept of Microbiology, University of B.C.).  spectrophotometer  Using a light  the absorbance of the purified O X - 6 Ig was  measured, and the protein (antibody) concentration was calculated to be 20.25 mg/ml.  The isolated islets were then incubated with  purified OX-6 Ig (at a concentration of 0.2 mg/ml) in 200 JJL 1 wells for 2 hours at room temperature.  After the primary incubation, the  islets were again washed by 2 complete volume exchanges with sterile PBS. A l l secondary incubations were carried out in duplicate, and in low light conditions.  The islets were incubated with each of two  RAMIg-BPD conjugates of different relative molecular ratios.  In  addition, the islets were also incubated with the "irrelevant" conjugate, with BPD alone, and with media alone, as controls.  The  secondary incubations were carried out for 2 hours, at room temperature.  The primary and secondary  are summarized in Table 2:  islet-antibody  incubations  page  24  Table 2: 1 ° Incubation  2 ° Incubation  (2 hr)  (2 hr)  OX-6 Ig (0.2 mg/ml)  RAMIg-BPD (1 : 6.5)  it  RAMIg-BPD (1 : 18) GA7s Ig-BPD  4.4  it  BPD alone  it  l l m M Ham's F-12 media alone  Photoactivation of BPD-bound Islets: Upon completion of the secondary incubation, the islets were  again washed with sterile PBS and resuspended in 11 m M glucose Ham's F-12 for light exposure.  The light source was a bank of four  fluorescent tubes (General Electric F20T12- cool white), and the spectrum of light emitted ranged from 300-750 nm.  The intensity of  the light was measured by a YS1- Kettering Model 65 RAdiometer and was 1.5 milliwatts per cm^.  The islets were exposed for 1 hour  at a distance of 11.0 cm from the light source, where temperatures were measured and did not exceed 25 C .  After the photoactivation  period, the islets were washed, resuspended in complete Ham's F-12 media and cultured overnight.  The following morning,  immunocytochemistry was performed on the treated islets to assess APC  depletion.  page  25  5) E V A L U A T I O N OF APC DEPLETION B Y IMMUNOCYTOCHEMISTRY: Immunocytochemistry was used to visualize Ia+ cells in cryostat sections of fresh, cultured and BPD-treated islets.  After a  thorough washing with HBSS to remove extraneous protein, 75-100 islets were deposited in polypropylene cassettes containing O C T medium, and were snap frozen with liquid N2.  The frozen cell block  was mounted in a cryostat microtome (-20 C), and 6 Jim islet sections were cut and mounted on glass slides.  The cryostat sections were  then fixed with dilute acetic acid and air dried, prior to rehydration with PBS and application of antibody.  A 2-layer technique of  antibody staining was used to visualize Ia+ cells:  first a (mouse)  anti-rat la M A b (OX-6 Seralab, 3.3 jig/ml) was applied and allowed to incubate with the cryostat sections for 1 hr at room temperature, or overnight at 4 C . fluorescein  After washing off excess primary antibody, a  indothiocyanate  (FITC) labelled (goat) anti-mouse  secondary Mab (Jackson Laboratories, 6.7  jig/ml) was applied and  allowed to incubate for 1 hr at room temperature.  After washing off  unbound secondary antibody, 10% glycerol and a coverslip were applied to the islet sections, which were then examined in the dark using a fluorescent microscope (Zeiss instruments).  With this  technique, 1-5 APCs could be identified within each fresh islet section, by their enhanced fluorescent characteristic dendritic morphology.  surface staining and A P C depletion was  qualitatively by a relative absence of fluorescent  cell  evaluated  enhancement  compared to fresh islet controls. 6) T R A N S P L A N T HISTOLOGY STUDIES: Approximately 100 fresh, cultured or BPD-treated SD islets were transplanted under the renal capsule of a W F recipient using a micropipette.  Twelve days later a recipient nephrectomy  was  performed, and the grafts were recovered, Bouins fixed, paraffin imbedded and stained with hematoxylin and eosin, then examined microscopically by a blinded observer, to determine engraftment or rejection had occurred.  whether  Rejection was determined  histologically by numerical grading of endocrine cell integrity (<+l  on  page  26  a scale of 0 to +4) and inflammatory cell infiltration (> +3 on a scale of +1 to +4), and by the absence of graft neovascularization. 7) C U L T U R E D ISLET ALLOTRANSPLANTATION OF STREPTOZOTOCINDIABETIC W F RECIPIENTS: These islets only. intravenous  experiments  were performed with  culture-modulated  W F recipients were rendered glucose intolerant by treatment  with streptozotocin,  75  mg/kg, and  maintained without insulin for a minimum of three weeks prior to allotransplantation, to insure that native 6 cell function and normoglycemia, would not return.  Hyperglycemic recipients were  then transplanted with approximately 1000 islets via the portal venous system.  fresh or cultured SD  Under ether anaesthesia, a  recipient laparotomy was carried out, and with the aid of the dissecting microscope, vascular control was obtained on a cecal mesenteric  vein.  A fine polyethylene catheter was used to cannulate  this vein, and a washed islet suspension in sterile HBSS was gently injected.  Transplant recipients were treated with a single  intraperitoneal dose of long acting insulin (9U/kg), for 4 days, and then plasma glucose determinations were commenced on Day 6, using a glucose analyser (Beckman II, Beckman Instruments). Allograft rejection was defined by consecutive determinations  greater  than 400  mg/dl.  plasma glucose  page  27  RESULTS: 1) ISLET IMMUNOMODULATION B Y TISSUE C U L T U R E : 1.1  A P C Depletion Experiments: Islet A P C depletion experiments  were performed to determine  the duration of culture necessary to remove la positive cells from SD rat islets as detected  by indirect fluorescent immunostaining.  Cryostat sections of freshly isolated rat islets were observed to contain between one  and five Ia+ cells per section.  With increasing  culture periods of four and seven days, a decrease in the number of staining cells per section was observed (Figure 1).  After a minimum  of 10 days in culture, la positive cells could no longer be demonstrated in islet cryostat sections (Figure 2). 1.2  Islet Allograft Histology Studies: Islet allograft histology studies were carried out to determine  the duration of pre-transplant culture necessary successful islet allografting.  to allow  consistently  Islet allografts cultured for periods of up  to 10 days prior to transplantation were uniformly rejected (Figure 3).  Four of 10 allografts cultured for 14 days prior to transplantation  demonstrated histologic engraftment, as did 4 of 5 allografts cultured for 21 days (Figure 4).  Allograft endocrine viability was  demonstrable by immunoperoxidase staining for insulin (see 5).  Figure  Table 3 summarizes the A P C depletion and allograft histology  studies.  page  28  Table 3: Duration of Tissue Culture (days) APC  Depletion:  0  4  7  10  14  21  yes  yes  yes  no  no  no  0  0  0  0  +4  +4  +4  +4  +4  +4  +1  +1  Neovascularization  no  no  no  no  yes  yes  # of Animals  5/5  5/5  5/5  5/5  4/10  4/5  Presence of Ia+ cells  ***************************************** Allograft Islet  Integrity  Mononuclear  1.3  Histology:  Infiltrate  In Vivo Allograft Function: Based on the finding that islet allografting was  histologically  successful  consistently,  when islets were cultured for 21 days prior  to transplantation, in vivo studies were carried out on treated,  hyperglycemic  streptozotocin  but otherwise immune-competent  recipients of fresh, 14 day and 21 day cultured SD islets.  WF Pilot in  vivo studies involved transplantation of islets under the renal capsule of the hyperglycemic recipient in an attempt to match the allograft histology  studies, however it was apparent that the volume  of islets required to effect euglycemia initially, could not undergo satisfactory of  revascularization at this location.  streptozotocin  Therefore, allografting  induced hyperglycemic recipients  was carried out  through the portal venous system, which allowed the islets to settle in the liver sinusoids, where they could easily acquire micro vascularization. Figure 6 shows the typical appearance of in vivo allograft rejection, after transplantation with uncultured islets.  There is  evidence of early graft function between day 6 to 10 post-transplant, followed  by persistent hyperglycemia heralding graft rejection.  Figure 7 depicts and graft  demonstrates function.  allotransplantation with 14 day cultured islets,  a similar phenomenon of graft rejection after early  page  29  In contrast, Figure 8 demonstrates 30 day function of 21 day cultured islet allograts  in streptozotocin-rendered  hyperglycemic  recipients. 2) ISLET IMMUNOMODULATION B Y PHOTODYNAMIC T H E R A P Y (PDT): 2.1  A P C Depletion Experiments: These experiments demonstrated selective depletion of APCs in  photoactivated islets which had been treated with the  specific  secondary RAMIg-BPD conjugate, at two relative molecular ratios (Figure 9).  In contrast, islets treated with either an irrelevant  secondary antibody conjugate (GA7sIg-BPD), BPD alone or media alone prior to photoactivation, all demonstrated preservation of islet APCs (Figure 10). 2.2  Transplant Histology Results: Syngeneic (3)  and allogeneic (4) renal subcapsular transplants  were carried out using islets which had been treated by anti-class IIspecific P D T . allogeneic  Subsequent histologic evaluation of both syngeneic and  grafts revealed complete replacement of graft by  lymphocytic infiltrate without identifiable endocrine tissue.  page  30  DISCUSSION: The aim of this thesis was to test Snell's "passenger theory, using a rodent pancreatic islet allograft model. this hypothesis,  leukocyte"  According to  allograft rejection is mediated by specialized donor  lymphoid cells called "antigen presenting cells" (APCs), which are carried passively into the host with the transplanted tissue.  Antigen  presenting cells consist of activated B and T lymphocytes, dendritic cells of Langerhans, macrophages and in some cases, capillary endothelial cells, and are characterized by their unique expression of class II M H C (la) antigen.  Within the immune-competent  allograft  recipient, APCs process and present foreign tissue antigen or "alloantigen" to histoincompatible responder lymphoid cells, initiating the allograft response.  thus  The M H C disparity that exists  between donor APCs and responder (host) T cells provides the stimulus for this antigen-specific  clonal immune response;  accordingly, the removal of APCs from the donor tissue prior to allotransplantation should prevent host recognition of Furthermore a permissive  alloantigen.  host environment should exist  indefinitely, provided no donor-identical APCs are introduced.  We  sought to test this hypothesis using two in vitro techniques of donor A P C depletion: directed  1)  in vitro culture, and 2)  photodynamic  monoclonal antibody-  therapy.  Isolated rat islets were subjected to variable periods of in vitro culture at specified conditions, then were examined with immunocytochemistry to determine necessary to deplete APCs.  the duration of culture  Although the technique used to evaluate  A P C depletion was qualitative, a clear trend of progressive A P C depletion with prolonged culture was observed.  Specifically, we  found that a culture period of 7-10 days was required for A P C removal from islet cryostat sections.  The mechanism of A P C  depletion by tissue culture is thought to be related to the  sensitivity  of lymphoid cells to local culture conditions including p02, pH and temperature. After determining that a 7-10 day period of culture was necessary  to deplete islet APCs, allogeneic transplants of fresh and  page  31  cultured Sprague Dawley (RTlu) islets were placed under the renal capsule of immune-competent Wistar Furth (RTla) recipients. Twelve days after transplantation, the allografts were removed and histologic  studies by a blinded observer employing strict criteria  (degree of mononuclear infiltrate, endocrine cell integrity and graft neovascularization) had occurred.  were  performed to determine whether  Utilizing this technique, we found that  rejection  allografts  transplanted either fresh or after periods of culture of 4, 7 and 10 days were all rejected.  In contrast, 4 of 10, 14 day cultured islet  allografts, and 4 of 5, 21 day cultured allografts demonstrated engraftment.  One explanation for the apparent disparity in culture  period necessary to deplete islet APCs versus that required for successful allografting is based on the observation that class II M H C antigen expression is an inducible phenomenon (80).  During the first  7-10 days of culture, expression of class II M H C antigen by the A P C may  be progressively down-regulated until antigen expression  below the limit of detection by immunocytochemistry. due to an absence  is  This could be  of lymphokine- (for example, gamma-interferon)  supported or stimulated class II M H C antigen expression by APCs in culture.  Thus, one could hypothesize that a culture "window" exists  during which APCs are viable but cannot be detected by immunocytochemistry because M H C antigen.  they have ceased to express class II  If the islets remain in culture for longer periods (21  days), the APCs die as a result of their relative sensitivity to the conditions of culture (pH, p02, cells.  temperature) compared to endocrine  Allogeneic transplantation of cultured islets during this  "window" period could result in host-lymphokine induced reexpression of class II M H C antigen by donor APCs, which permits a donor A P C mediated allograft reaction to occur.  Alternatively, one  can hypothesize that elimination of donor APCs is not by itself, responsible for prevention of the allograft reaction; vitro culture must impose other changes  therefore in  on tissue (perhaps  alteration of alloantigen or irreversible inhibition of A P C function), which permit prolonged allograft survival after a requisite period of pre-transplant culture.  Recent investigations  have demonstrated a  potential role for islet treatment regimes which alter or block donor  page  32  potential role for islet treatment regimes which alter or block donor specific M H C class I antigen.  Stock et al (81) demonstrated that  whole mouse islet pretreatment with anti-class antibody blocked the generation  I monoclonal  of allo-specific  cytotoxicity  target cells following a mixed islet-lymphocyte This permits speculation  that culture specific  against  co-culture period. immunomodulation  may occur through depletion or alteration of class I M H C antigen, which may also explain the observed disparity in culture time required to deplete APCs versus that necessary for  successful  allotransplantation. Of perhaps greatest significance  was the demonstration  that  islets were capable of in vivo function for at least 30 days when transplanted  into  non-immunosuppressed,  following 21 days of in vitro culture.  allogeneic  recipients  This was shown in both W F  recipients of 21 day-cultured SD islets that had been rendered glucose intolerant by treatment with streptozotocin.  In contrast, islet  allografts cultured for 14 days prior to transplant (2 animals), or without any period of pre-transplant culture (1 animal) were all rejected.  These  transplant recipients  hyperglycemia following a brief (7-10 manifest  demonstrated  persistent  day) period of graft function  by euglycemia, which correlates  temporally with the period  of time necessary to mount an ablative immune  response.  The implications of these experiments are significant:  using an  in vitro islet culture technique of immunomodulation in a rodent model, we have demonstrated successful without  allogeneic host  both histologically  and functionally  transplantation across a major M H C barrier,  immunosuppression.  The second set of experiments  were done to test photodynamic  therapy (PDT), as a possible immunomodulatory modality in islet transplantation.  P D T has not yet found experimental application in  transplantation, though its utility in the treatment  of malignancy,  namely tumors of the bladder, esophagus and bronchus is currently being evaluated in phase III comparative, controlled clinical trials (82).  The success of P D T relies on the delivery of the porphyrin  molecule to the target cell (in this case, the islet APC), by a specific carrier monoclonal antibody.  Once bound to its cellular target, the  page  33  porphyrin molecule is photoactivated by U V light causing emission of singlet oxygen which results in lipid peroxidation, cell membrane distruption and ultimately cell death. Our technique of A P C photoablation by P D T required a primary incubation of islets with OX-6 (an anti-class II M H C monoclonal) followed by incubation of a specific secondary monoclonal antibody (RAMIg) which had been conjugated to BPD.  After photoactivation,  the islets were cryostat sectioned and immunostained for class II M H C + cells.  The depletion of APCs by this technique was specific,  and seen only in islets which had been exposed to the specific secondary antibody-BPD conjugate prior to photoactivation.  In  contrast, islets treated with an irrelevant porphyrin-antibody conjugate, or porphyrin alone showed relative A P C preservation, at least as detected by fluorescent immunostaining.  When specifically  treated islets were transplanted under the renal capsules of immune-competent syngeneic  and allogeneic  hosts (3 and 4  transplants, respectively), and the grafts removed at 12 days, there was histologic evidence of both acute and chronic inflammation without signs of graft survival.  The failure of even syngeneic grafts  permits speculation that perhaps the rigors of photodynamic therapy injured the islets irreversibly, so that the histologic appearance of the harvested  grafts  represented  a non-specific inflammatory  response to necrotic tissue, rather than a specific immune response. This hypothesis  seems the most probable, given the altered  fluorescent microscopic appearance of BPD-treated islets compared to their fresh and cultured counterparts (compare figures figures 9 and 10).  1 and 2 with  Freshly isolated islets and those maintained  viably in tissue culture have a typical "honeycomb" appearance, based on a faint, fluorescent outline of individual endocrine cells.  In  contrast, islets undergoing photodynamic therapy with BPD did not demonstrate  the same individual cell preservation when examined  with fluorescent microscopy, suggesting  a non-specific cellular injury.  It is quite likely that further experiments such as immunostaining of treated islets for insulin production would help to clarify the viability and functional status of the endocrine cells after PDT. On the other hand, based on our own experience with failed syngeneic  page  34  transplantation of freshly isolated Wistar Furth islets into immunecompetent  recipients,  there is sufficient  and through personal communications  (83),  evidence to suggest that the Wistar Furth rat strain  is not highly inbred.  It is therefore possible that  minor M H C  incompatibilities exist within the strain which are responsible  for  apparent "syngeneic" graft rejection, and that the rejection of all three "isografts", (as well as all four allografts) following islet treatment by M H C class II specific P D T , merely reflects the inability of donor A P C depletion alone, to prevent the allograft reaction. Despite our inability to demonstrate a facilitation of transplantation with P D T , it remains an exciting  allogeneic  prospective  application in the field of immunomodulation on the basis of its cellular specificity  (by virtue of the carrier monoclonal antibody),  and rapidity of action.  Clearly, experimental refinements  necessary to maintain specificity  are  of target cell destruction  without  injury to surrounding parenchymal cells. In conclusion, the fundamental stumbling block to tissue transplantation, whether it be vascularized, solid organ or nonvascularized, cellular (such as pancreatic islet), is allograft rejection. Circumvention of allograft rejection requires either host immunosuppression, reduction of donor tissue immunogenicity, the two in combination.  Anti-rejection strategies in pancreatic islet  transplantation have focussed  on in vitro techniques  APCs or "passenger leukocytes" from the donor tissue. has described the evaluation of two techniques tissue treatment  allogeneic  which eliminate This thesis  of in vitro donor  , namely pre-transplant tissue culture and  monoclonal antibody guided photodynamic therapy. techniques  or  demonstrated  selective A P C depletion,  transplantation was possible  Although both successful  only after islets were  cultured for a period of time significantly longer than that necessary to deplete APCs.  This suggests that donor A P C depletion alone cannot  prevent allograft rejection, and that in vitro culture must reduce donor tissue immunogenicity allogeneic permitted.  by some other mechanism, such that  transplantation into an immune-competent  host is  page  35  Bibliography: 1.  Loeb L . The Biological Basis of Individuality.  Charles C Thomas,  Springfield IL, 1945. 2.  Murphy JB. 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Personal communication, Dr R McMaster.  46:  Figure la:  Figure lb:  Figure 1: Fluorescent micrographs of fresh (la) and 7 day cultured (lb) Sprague Dawley islets after cryostat sectioning and 2 step fluorescent immunostaining for la + cells (arrows); (x 400 - l a , x 200 -lb).  page 42  Figure 2 : Fluorescent micrograph of 14 day cultured Sprague Dawley islets after cryostat sectioning and 2 step fluorescent immunostaining for la + cells; (x 400). Note absence of fluorescent cellular staining (compared to figure 1).  page 43  Figure 3: Rejected, fresh Sprague Dawley islet allograft under renal capsule of Wistar Furth recipient, harvested 12 days after transplantation; (H & E stain, x 100). Note extensive mononuclear infiltrate between kidney capsule (KC) and kidney (K), which has completely replaced graft.  page 44  Figure 4: 14 day cultured Sprague Dawley islet allograft under renal capsule of Wistar Furth recipient, harvested 12 days after transplantation; (H & E stain, x 100). Note integrity of endocrine cells of graft (G), between kidney capsule (KC) and underlying kidney (K).  page 45  Figure 5: Engrafted, 14 day cultured Sprague Dawley islet allograft under renal capsule of Wistar Furth recipient, stained with immunoperoxidase for insulin- confirming endocrine activity of transplanted islets after a period of in vitro culture; (H & E stain, x 100). K C = kidney capsule, K = kidney.  page 46  Figure A plot Wistar via the mg/dl)  6: of plasma glucose vs time in an immune competent Furth recipient of a fresh Sprague Dawley islet allograft portal vein (PV). Persistent hyperglycemia (> 400 signifies graft rejection.  page 47  Figure 7: A plot of plasma glucose vs time in immune competent Wistar Furth recipients of 14 day cultured Sprague Dawley islet allografts via the portal vein (PV). Early graft function has led to persistent hyperglycemia, signalling graft rejection.  page 48  Figure 8: A plot of plasma glucose vs time in immune competent Wistar Furth recipients of 21 day cultured Sprague Dawley islet allografts via the portal vein (PV), demonstrating 30 day in vivo allograft function.  page 49  Figure 9: Fluorescent micrograph of cryostat-sectioned Sprague Dawley islets immunostained for la + cells, following photodynamic therapy (PDT) with specific RAMIg-BPD conjugate; (x 400). Note absence of fluorescent cellular staining compared to fresh, untreated islets (figure la), and islets which have undergone PDT using an irrelevant antibody-BPD conjugate (figure 10). (A = artefact)  page 50  Figure 10: Fluorescent micrograph of cryostat-sectioned Sprague Dawley islets immunostained for la + cells, following photodynamic therapy (PDT) with an irrelevant antibody-BPD conjugate; (x 400). Note preservation of la + cell, in contrast to figure 9.  


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