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GIDEON, Jr., Leonard Albert, 1943- IMMUNOLOGICAL PARAMETERS OF RENAL TRANSPLANTATION IN THE CANINE.

The Ohio State University, Ph.D., 1972 Microbiology

!

University Microfilms, A XEROX Company, Ann Arbor, Michigan j

.... _ 1

THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED IMMUNOLOGICAL PARAMETERS OF RENAL

TRANSPLANTATION IN THE CANINE

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

by

Leonard Albert Gideon, Jr., B.S., D.V.M., M.S.

*****

The Ohio State University 1972

Approved by

Advisor epartment of Microbiology 1/ PLEASE NOTE:

Some pages may have

indistinct print.

Filmed as received.

University Microfilms, A Xerox Education Company ACKNOWLEDGEMENTS

To Dr. John H. Wallace, advisor and friend, I wish to offer my greatest appreciation for his constant encouragement, guidance and sincere interest. Dr. Wallace, you are a rare combination of a thoughtful person and scholar.

I wish to thank Drs. Ronald St. Pierre, Matthew Dodd and Frank

Chorpenning for serving on the examining committee and offering helpful suggestions in the preparation of the manuscript. An especial appre­ ciation is extended to Dr. St. Pierre for the personal counseling he provided and for the preparation of the mounted slides for histological examination.

I wish to thank Drs. James Madura, James Cerilli, Leroy Johnson,

Jay Stein, Milton Wyman, Alden Stilson and Alton Wilson for their guidance, encouragement and/or generosity in providing facilities and materials during the course of this research project.

I am deeply grateful to the National Institute of Health and

Merilyn C. Hiller, Hematology Program Director, National Institute of

Arthritis and Metabolic Diseases, for supporting me on a Post Doctoral

Fellowship for the past three years.

Finally, I wish to thank my wife, and my parents for their encouragement and prayers throughout the years. VITA

December 27, 1943 Born, Portland, Oregon

1966 ...... B.S. degree, Texas A and M University, College Station, Texas

1967 ...... D.V.M. degree, Texas A and M University, College Station, Texas

1968-1969 . . . American Veterinary Medical Assoc. Fellow

1968-1971 . . . Teaching Associate, Department of Veterinary Surgery, The Ohio State University

1969 ...... M.S. degree, The Ohio State University, Columbus, Ohio

1969-1972 . . . National Institutes of Health Post Doctoral Fellow

PUBLICATIONS

1. Cerilli, G. James and Gideon, Leonard. "Successful Long Term Inhibition of Xenograft Rejection." Surgical Forum, 10, 1969.

2. Cerilli, James, Gideon, Leonard and Hattan, Dorothy. "Effect of Immunosuppression and Cellular Antigen on Xenograft Survival.” Transplantation Proceedings 2, 1970, p. 516.

FIELDS OF STUDY

Major Field: Veterinary Medicine

Veterinary Surgery. Dr. Leroy Johnson

Immunology. Dr. John H. Wallace TABLE OF CONTENTS

page AC KNOWLEDGE NE NT S ...... ii

VITA ...... iii

LIST OF P L A T E S ...... v

LIST OF FIGURES ...... vii

LIST OF TABLES ...... ix

Introduction ...... 1

Review of the Literature ...... 6

Experimental Procedure ...... 38

Results ...... 60

D i s c u s s i o n ...... 139

Summary ...... 155

BIBLIOGRAPHY ...... 157 LIST OF PLATES

Plate Page

1. Rabbit Anti-Coyote Serum Reacted with Dog and Coyote Sera in the Ouchterlony Te s t ...... 61

2. Rabbit Anti-Dog Serum Reacted with Dog and Coyote Sera in the Ouchterlony T e s t ...... 62

3. Rabbit Anti-Coyote Serum Reacted with Six Different Coyote Sera in the Ouchterlony T e s t ...... 63

4. Rabbit Anti-Dog Serum Reacted with Six Different Dog Sera in the Ouchterlony Test ...... 64

5. Rabbit Anti-Dog Serum Unabsorbed (a) and Absorbed with Dog (b) and Coyote (c) Sera and Reacted with Dog and Coyote Sera in the Ouchterlony T e s t ...... 66

6. Immunoelectrophoresis of Dog and Coyote Sera Reacted with Rabbit Anti-Coyote Serum ...... 67

7. Immunoelectrophoresis of Dog and Coyote Sera Reacted with Rabbit Anti-Dog Serum ...... 68

8. Immunofluorescense of Erythrocytes from a C-Antigen Positive Dog ...... 123

9. Cortical Biopsy of a Coyote Kidney Showing Normal Parenchyma ...... 127

10. Biopsy from a Rejected Coyote Kidney in the No-Treatment G r o u p ...... 128

11. Biopsy from a Rejected Coyote Kidney in the No-Treatment G r o u p ...... 129

12. Biopsy from a Rejected Dog Kidney in the HADLG-Treated G r o u p ...... 131

13. Biopsy from a Rejected Coyote Kidney in the HADLG-Treated Group ...... 132

v LIST OF PLATES CONTINUED

Plate Page

14. Biopsy from a Rejected Coyote Kidney in the HADLG and Antigen-Treated G r o u p ...... 133..

15. Biopsy from a Rejected Coyote Kidney in the Antigen- Treated Group ...... 135

16. Biopsy from a Rejected Coyote Kidney in the Homologous ALG-Treated Group ...... 136

vi LIST OF FIGURES

Figure Page 1. Horse Anti-Dog Lymphocyte Serum: Progression of Antibody Titer with Immunization ...... 70

2. Horse Anti-Dog Lymphocyte Serum: Serum Absorption with Dog Erythrocytes ...... 71

3. Progression of Lymphocyte Cytotoxic Antibody Production with Immunization ...... 79

4. Progression of Lymphocyte Cytotoxic Antibody Production with Immunization ...... 80

5. Dog (75) Leukocyte Response to 3052 Anti-3051 Anti­ lymphocyte Globulin Administration ...... 81

6. Dog (99) Leukocyte Response to 3041 Anti-3049 Anti­ lymphocyte Globulin Administration ...... 82

7. Leukocyte Response of the Dogs in the No-Treatment Group ...... 95

8. Leukocyte Response of the Dogs in the HADLG-Treated Group ...... 97

9. Leukocyte Response of Dog 9974 to HADLG Treatment and Transplantation with Dog 3049 K i d n e y ...... 102

10. Leukocyte Response of Dog 0P-10 to Homologous ALG Treatment and Transplantation with Coyote 12 Kidney 108

11. Leukocyte Response of Dog A-l to Homologous ALG Treatment and Transplantation with Coyote 12 Kidney 110

vii LIST OF FIGURES CONTINUED

Figure Page 12. Leukocyte Response of Dog 3051 to Homologous ALG Treatment and Transplantation with Dog 3052 Kidney . . . 113

13. Leukocyte Response of the Dogs in the HADLG and Antigen Treatment Group ...... 115

14. Leukocyte Response of Dog 3044 to Lymphoid Antigen Pretreatment and Transplantation with Coyote 6 Kidney . . 118

viii LIST OF TABLES

Table Page 1. Horse Antibody Against Dog Lymphocytes: Characteristics of the Ammonium Sulfate Precipitated Product ...... 73

2. Reactivity of Horse Anti-Dog Lymphocyte Serum in the Direct Hemagglution Assay ...... 74

3. Horse Antiserum Against Dog Lymphocytes ...... 76

4. Rabbit Antisera Against Dog and Coyote Lymphocytes . . . 77

5. Dog and Coyote Antibody Against Dog and Coyote Lymphocytes: Characteristics of the Ammonium Sulfate Precipitated Product ...... 84

6. Reactivity of Dog and Coyote Antisera Against Dog and Coyote Lymphocytes ...... 85

7. Dog WPUP Antiserum Against Coyote 12 Lymphocytes .... 86

8. Dog WPUP Antiserum Against Coyote 12 Lymphocytes .... 87

9. Coyote 12 Antiserum Against Dog 3051 Lymphocytes .... 89

10. Dog 3052 Antibody Against Dog 3051 L y m p h o c y t e s ...... 90

11. Dog 3052 Antiserum Against Dog 3051 Lymphocytes ...... 92

12. Dog 3041 Antiserum Against Dog 3049 Lymphocytes ...... 93

13. Dog Antibody Against Horse Globulin: Indirect Hemagglu­ tination Assay 100

14. Histocompatibility Typing ...... 103

15. Histocompatibility Typing ...... 106

16. Histocompatibility Typing ...... 109

ix LIST OF TABLES CONTINUED

Table Page 17. Histocompatibility Typing ...... 112

18. Summarized Survival Data: Renal Transplants ...... 119

19. Antisera from Dogs with Coyote Renal Grafts: Lymphocyte Cytotoxic Assay ...... 120

20. Antiserum from Dog 3044 Grafted with Coyote 6 Kidney . . 124

21. Antisera from Dogs with Coyote Renal Grafts: Direct Hemagglutination Assay ...... 125

22. Distribution of the Canine Blood Group Antigens .... 137

23. Incidence of Combinations of Blood Group Antigens in the Canine ...... 138

x INTRODUCTION

The most serious limiting factor in clinical transplantation at this time is the supply of suitable donor organs. The fundamental immunologic considerations in transplantation, although incompletely understood, are being managed with increasing success due in large measure to histocompatibility testing and to improved regimens of recipient immunosuppression.

Wide application of organ transplantation will require an abun­ dant source of suitable organs. The use of living donors raises serious ethical and legal problems and is of benefit only in the donation of an organ in a paired organ system. Cadaveric organs, even if free from disease, are sometimes found to have sustained serious agonal or post mortem damage.

Improved experimental methods of in vitro (Abbuna, ei al,., 1972) and (Cooperman, et al.., 1971) and in vivo (Benjamin and Sell, 1972) organ preservation have given insight as to means of reducing organ wastage. Collaborative organizations such as Eurotransplant (Van

Rood, 1971) have already done much to promote a more effective utili­ zation of available organs through widespread exchange of information concerning prospective recipients and potentially available donors.

However, if organs from animals could be used in man, or if human organs could be preserved for prolonged periods in animals prior to subsequent return to a human recipient, the problem of supply versus demand could be more effectively handled in the future. Most ethical and legal problems that plague the use of human donated organs would also be diminished.

The field of cross species transplantation (xenografting) was explored early in this century. Early attempts to place tissue from a widely divergent species into man uniformly failed (Princeteau,

1905) (Neuhof, 1923) and (Avarmavici, 1924). Failure to achieve long term survival of whole organ grafts even in an intra-species trans­ plant (allograft) led to a waning of interest in clinical application of organ grafting.

The studies of Medawar (1944, 1945) established that the rejec­ tion of skin allografts was basically an immunologic reaction. Later this same mechanism was shown to be involved in the rejection of whole organ grafts (Dempster, 1953) and (Simonson et aj^., 1953).

Interest in clinical allografting was renewed by a report on nine renal allografts performed by Hume (1955). This report provided the background for subsequent development in the field of renal trans­ plantation.

The improving results in allotransplantation served to highlight the difficulty of the procurement of suitable donor organs. In instances in which patients were terminally ill and without a suitable organ donor, xenografts were attempted. Observations on the first chimpanzee-to-human transplant in 1963 (Reemtsmal, e£ aj,.., 1964a) renewed interest in clinical renal xenografting by demonstrating that such grafts could function in some respects like allografts. The immunologic aspects of renal xenografting have been studied extensively by DeWitt (1965) in chimpanzee-to-man grafts, and by Kirk­ patrick and Wilson (1964) in baboon-to-man grafts. Of the sub-human primates, the chimpanzee is most like the human antigenically and thus serves as the most feasible donor of organs to humans in a xenograft system.

Extensive studies of sera from patients having received a chim­ panzee kidney (DeWitt, 1965) and of sera from volunteers immunized with chimpanzee tissue (DeWitt eiaJL., 1968) and visa versa (Metzgar,

1965) have revealed both allogeneic and xenogeneic antibody specifi­ cities. In addition to partial sharing of leucocyte transplantation antigens, the chimpanzee can also be typed as human blood group A or

0 on the basis of response with anti-A and anti-B typing sera

(Wiener and Moor-Jankowski, 1963). Other studies suggesting close relationship between chimpanzee and man involves serum proteins, hemoglobin structure and DNA structure (Goodman, 1970). Analogous studies have demonstrated similarities in renal function (Gagnon,

1957).

Animal models for studying the rejection phenomenon in xeno­ grafting should include paired species that simulate the antigenic relationship that exists between the human and the chimpanzee. The experimental studies of Perper and Najarian (1966a, 1966b, 1967) have done much to clarify the importance of genetic disparity in xeno­ grafting.

In this present study, the domestic dog (Canis familiaris) and and the coyote (Canis lantrans) viere considered as a xenograft model.

The coyote and dog reportedly belong to closely related species

(Leone and Wiens, 1956) and (Jones, 1969). In a very limited study of the relationship between the serum proteins of these animals, it was reported that rabbit antibody prepared against dog serum cross reacted with coyote serum in the precipitation reaction. Homologous reactivity was rated at lOC^ and cross reactivity at 54%. Similar results were obtained with a rabbit anti-coyote serum reagent. With the exception of the unpublished work of Bull and his associates

(1971) concerning shared erythrocyte antigens, there is no other

information available about the antigenic relationships between the dog and the coyote.

Consequently, we were interested in determining something of these relationships before the initiation of extensive studies con­ cerned with the survival of renal grafts between these animals. The

initial approach was to examine the findings of Leone and Wiens (1956) with serum proteins. However, since cellular antigens are of primary

interest for transplantation studies, the lymphocyte antigens of the

two species'will receive the most attention here.

Antibody against cellular antigens were produced in the rabbit,

horse, dog, and coyote by lymphoid cell inoculation and skin graft

immunization. Additional antisera against coyotes were generated in

dogs receiving coyote renal grafts. These antisera were employed in

serologic tests for determining the antigenic relationships between

the dog and the coyote. In addition, host immunologic and clinical response to the transplant was studied during the course of concomi­ tant immunosuppressive attempts. The immunosuppressive measures employed have included the administration of heterologous or homolo­ gous antilymphocyte globulin and the injection of solubilized cellular antigen of graft donor origin; the latter procedure was for the pur­ pose of attempting the induction of immunologic enhancement. REVIEW OF THE LITERATURE

Historical Background

Transplantation of tissue for the replacement of non-functional

parts due to disease, age or injury has stimulated the imagination of

man for many centuries. Reports of transplantation prior to the

nineteenth century were concerned primarily with skin grafting. The

first clinical report in modern medical literature of a successful

skin transplant was by Bunger (1823) who reconstructed part of a

woman's nose by a free graft from the thigh. Reverdin (1869) des­

cribed his success in covering granulating surfaces with small pieces

of epidermis. Ivanova (1890) reported the successful use of skin

from stillborn infants in the treatment of an aged burned patient.

Other reports of similar results with split-thickness (Oilier, 1872)

and full-thickness (Lav/son, 1872) skin grafts were reported. No dis­

tinction was made, at this early period, between the behavior of skin

grafts transplanted from another portion of the patient's own body

(autograft), those taken from another individual of the same species

(allograft), and even on occasions from another species (xenograft)

(Reverdin, 1872).

Schone (1912) was probably the first to suggest that allografting

was usually unsuccessful, and he coined the term "transplantation-

simmunitat” to explain the phenomenon suggesting that it was the result of some form of immune response. Lexer (1914) was never

successful in his attempts at allografting and suggested that the

result of scar substitution was being observed by others.

Shawan (1919) described a series of observations in which allo­

grafts obtained from donors of the same erythrocyte group as the

recipient appeared to persist longer than those from donors mis­

matched with the recipient. He concluded that there must be a rela­

tionship between successful skin grafting and compatibility for

erythrocyte antigens. A more significant observation was recorded by

Emile Holman (1924). He described two cases of repeat skin grafting

in children using the same donor. In both cases he observed an

accelerated 'melting-away* of the second grafts while a graft from a

third party donor remained undisturbed. He considered that his

observation strongly supported the immune concept of Schone (1912).

This report went virtually unnoticed and the significance of using

the same donor was not fully appreciated until Gibson and Medawar

(1943) reported an almost identical set of observations.

Organ transplants with their associated technical difficulties

were not attempted until the first decade of this century. Emerich

Ullman (1902) reported technical success with both autogenous and

homologous kidney transplants in dogs. He also gave reference to a

dog-to-goat transplant in an experimental study of renal grafting.

That same year he transplanted a pig kidney into a woman. He des­

cribed the failure of the kidney to function as a technical

difficulty (Ullman, 1914). Several other unsuccessful attempts to transplant kidneys from animals to man were made. Princeteau (1905) reported inserting slices of rabbit kidney into a nephrostomy in a child with renal insufficiency. In the following year, Jaboulay (1906) used vascular anastomosis in the transplantation of xenografts from a pig on one occasion and a goat on another into the antecubital spaces in two patients. Early failure was attributed to vascular thrombosis. The first non-human primate-to-man renal graft, performed by Unger (1910), was likewise without success. Neuhof (1923) grafted the kidney of a lamb into a patient when he was unable to procure a human donation.

The patient died 9 days later; however, Neuhof was encouraged by thinking that the graft did function for a while.

Further work in experimental organ grafting was done at the Mayo

Foundation by Dederer (1919; 1920). He reported a 26 day survival of a renal graft between canine liter mates. This work was extended by

Williamson (1923; 1926). In two instances, he performed xenografts using goats as donors and dogs as recipients. Each recipient died within a matter of minutes of what he interpreted as being an acute anaphylactic reaction. He was interested by the considerable varia­ tion in survival times seen in his own work and those reported by others. He postulated that such variations were the result of genetic relationship between donor and recipient. A year later, Bauer (1927) reported a successful and permanent skin transplant from one twin to another.

The basic concepts of the reaction of an unrelated host to transplantation of tissues from another individual were formulated by investigators in the field of tumor research. Ehrlich (1906) deve­ loped the "athrepsia" theory in which he supposed that the lack of a special nutritive material was the cause of graft death in tumors he transplanted between mice and rats. He observed that a mouse tumor if transplanted into the rat would grow for about eight days and then die. On transfer back to the mouse, however, it survived and after another eight days could be reimplanted in another rat where it would again remain viable for approximately 8 days before dying.

A second hypothesis, that of immunity, was advanced by Bashford,

Murray, and Haaland (1908) and by Russell (1912). They observed that once a rat destroyed a mouse tumor that had survived for 8 or 9 days, a second mouse tumor implanted into the same rat was destroyed in 3 to 4 days. From these findings they postulated that the rat had developed an immunity to mouse tissue as a result of the first trans­ plant and was able to destroy a second transplant more rapidly.

Murphy (1912), working with the Rous chicken sarcoma, observed that the tumor grew well in both the duck and pigeon embryo, but would not survive in the adult of either species. He similarly demonstrated that the rat Jensen tumor would survive for over six weeks by serial passage in the chicken embryo as compared with a survival time of 3 days in an adult of the same species. Murphy concluded, "This proves beyond doubt that the mammalian cells are able to utilize the food supplied by the avian embryo. Whether this phenomenon is dependent solely on this factor or whether the absence of a defense mechanism in the embryo plays the more important part is a subject which is 10 being studied Murphy (1914a) was also able to demonstrate that the prolonged survival of both homologous and heterologous tissue in the chick embryo could be reversed by the inoculation of adult fowl lymphoid suspension (both chicken spleen or bone marrow).

As a further extension of these observations, Murphy (1914b) used low doses of radiation and subcutaneous administration of benzol to modify the rejection process and did observe a prolonged survival of mouse sarcoma transplanted to rats receiving such treatment. It was well known at the time that roentgen rays given in small doses affects first and most pronouncedly the lymphoid system. These findings led

Murphy to conclude that the mechanism responsible for the destruction of a transplant was contained in a cell common to l) the transplanted lymphoid tissue, 2) the reaction around a homologous tissue trans­ plant undergoing rejection, and 3) the reaction around a foreign transplant in the embryo supplied with adult lymphoid cells. He identified the cell as the small lymphocyte.

The concept that allograft rejection was primarily a systemic immune response was not universally accepted as late as 1944. Loeb

(1944) did not accept the immune response theory because he was unable to demonstrate accelerated second-set graft rejection in his own work. However, he did not employ repeated allografts from the same donor. Loeb maintained that a host reacted in a purely local way against homotransplanted tissue. He conceived that the cellular infiltrate was caused by individuality differentials of host and transplant. Loeb's studies were histological interpretations on 11 pieces of kidney that had been implanted subcutaneously. Auto­ implants likewise showed cellular infiltration and architectural disentegration.

It remained for Medawar (1944, 1945, 1946a) to show for the first time the accelerated rejection reaction resulting from repeated graft­ ing from the same donor. This interest had been stimulated by a series of clinical observations in a burned patient who received successive grafts from the same donor (Gibson, 1943). Using a hetero­ geneous rabbit model, Medawar demonstrated a high degree of specifi­ city in a second-set response towards the skin of the rabbit that provided the immunizing graft. It was also evident that varying degrees of reactivity might be elicited against the graft of a third party donor indicating to Medawar a sharing of some tissue antigens.

He described the allograft destruction as an acute inflammatory reaction, with massive invasion by mononuclear cells of the host. In a subsequent paper (Medawar, 1946b), he demonstrated that the intra- dermal injection of foreign homologous leukocytes conferred a typical immunity towards skin later grafted from the leukocyte donor. He con­ cluded that leukocytes share antigens in common with the antigens of the skin.

Medawar’s evidence of the involvement of a systemic immune response in the rejection of skin allografts gave rise for a new critical evaluation of the nature of renal allograft failure. Simon- sen and associates (1953) and Dempster (1953) were responsible for first demonstrating the involvement of the systemic immune response in renal allograft rejection. 12

Dempster (1953) reviewed the literature on renal allografting and concluded that no success could be shown in either human or animal studies, and further, the biological mechanism of graft failure was not agreed upon. He proposed the use of a renal allograft model to lend support to the immunological basis of allograft rejection.

First-set and second-set grafts were placed by primary vascular anastomosis in the necks of 85 dogs. He described the lymphocyte and plasma cell infiltration of first-set renal grafts and demonstrated the accelerated rejection phenomenon of second-set grafts from the same donor with the accompanying interstitial edema and free hemorr­ hage. Dempster concluded that a systemic humoral immune response on the part of the host was predominately responsible for the graft rejection. He thought that the mononuclear cells seen in the parenchyma of the renal graft were of graft origin-— the cells having differentiated into lymphoid cells from reticuloendothelial cells of donor origin residing in the graft. Thus, the total mechanism of graft disentegration was the result of two reticuloendothelial systems pitted against one another.

Immunological Considerations

Abundant evidence is now available to implicate an immunological barrier as the chief impediment to successful transplantation. The clearest evidence that resistence to allografts is acquired and not ready made is that the survival time of a skin allograft varies inversely with the quantity of tissue that is grafted (Medawar,

1944). Medawar demonstrated in rabbits that an eight-fold reduction 13 of the area of skin that is transplanted in first-set fashion will increase the median survival time of allografts by about one-half.

The fact that a dosage relationship exists at all unseats the hypo­ thesis that resistance to allografts is ready made.

The allograft reaction has three properties which identify it beyond reasonable doubt as an immunological reaction. The first of these is the existence of the second-set graft destruction phenomenon which is like the secondary response of a host to a protein antigen.

Secondly, the existence of a phenomenon analogous to the passive transfer of immunity can be demonstrated. Mitchison (1954) showed that lymphoid cells expressed from the regional nodes of a mouse which had reacted against a local solid tumor allograft have the power to confer sensitivity upon a normal mouse. The second mouse, the recipient of the first mouses' activated lymphoid cells, behaves as if it had itself received and rejected the tumor graft beforehand, for when challenged by an allograft of the same tumor, it destroys it

2 or 3 times more quickly than would otherwise have been the case.

These same observations of adoptive immunity (passive transfer of competent cells) have been made using a skin graft model by Billing- ham and associates (1954, 1956a). These authors could not transfer immunity by means of serum from the same donor. This supported the idea that graft rejection was a cell-mediated reaction similar to delayed hypersensitivity. A comparative histological study of first- set skin graft rejection and various examples of delayed hypersensi­ tivity reactions had been made by Waksman (1964) with the conclusion that they were similar. In the studies on the cellular transfer of cutaneous sensitivity to certain bacterial substances in man,

Lawrence (1955) found that, under appropriate conditions, sensitivity of the delayed type could be transferred not only with whole viable cells from sensitive donors but also with cell-free leukocyte extracts. It has since been found that sensitivity to skin allografts in man (Lawrence, et al.. 1960) and guinea pigs (Powell, et al.. 1964) can be transferred in a similar fashion. In some way the subcellular fraction sensitizes cells of the recipient. These experiments show that transplantation immunity is transferable and suggest that the regional lymphnodes are the principal anatomic seat of reaction against a local homograft which establishes lymphatic connections in the host.

The third property that identified the allograft reaction as an immunological response is the fact that reactivity against allografts can be prevented from developing by taking advantage of the principle of immunological tolerance which is known to apply to other and entirely orthodox immunological systems.

Owen (1945) discovered that erythrocytic chimerism occurred among dizygotic tv/in cattlej in other words, each twin had a proportion of erythrocytes of the type proper to the other. This was explained by an interchange of hematopoietic stem cells through vascular anasto­ moses in the fetal placentas. Not only were the blood cells mutually acceptable but also grafts of skin (Billingham, e£ al.. 1952) and

(Anderson, et al.. 1951). Burnet and Fenner (1949) explained this observation by a postulate of the greatest theoretical importance.

They suggested that an animal fails to react against antigens with 15

which it comes into contact in embryonic life. The normal teleolo-

gical value of such a failure would be to avoid reactivity against

the animal's own antigens. A similar inertia might, they suggest, be

shown toward antigens introduced into the embryo from elsewhere —

either naturally or artificially.

The first successful attempt to induce immunological tolerance

was made by Billingham, Brent and Medawar (1953). Spleens were

removed from adult CBA mice and a single cell suspension made of them.

Approximately 5 x 10^ of these cells were injected, intravenously and

intraperitoneally, into A-line mice within 24 hours of birth. When

the injected A-line mice were 6 to 8 weeks old, the great majority

were found to accept CBA skin allografts for periods extending far

upwards of 50 days. Normally a skin allograft transplanted from an

adult CBA donor to a normal adult A-line recipient was invariably

destroyed within 12 days.

Besides the consistent observation that lymphoid cells were ever

present in histological sections of rejected grafts, other circum­

stantial evidence that small lymphocytes were involved at some stage

in the reaction to allografts comes from procedures which prevent the

normal development of lymphoid tissue or remove small lymphocytes from

the animal. Animals subjected to neonatal thymectomy showed in later

life a reduction in the number of small lymphocytes which were nor­

mally present in the lymph nodes, spleen and blood; and this defi­

ciency was associated with a severe impairment of the ability to re­

ject allografts and even xenografts (Good, e£ al.. 1963) and

(Miller, 1962). Grafts of neonatal thymus and injections of spleen 16 and lymph node cells from adult syngeneic donors restored the immuno­ logical responsiveness of thymectomized animals and also reconsti­ tuted their lymphoid tissue (Dalmasso, et al.. 1963), (Miller, 1963) and (Parrott and East, 1964). Humphrey and associates (1964) demon­ strated that while neonatal thymectomy greatly depressed cell-mediated reactions, like the allograft response, it left relatively intact the humoral-antibody forming capabilities of the host, at least to certain antigens.

The mechanism of the thymic influence over the "thymus dependent” cell population is not clear. It may be that direct processing by passage through the thymus, direct contact with thymus-derived cells elsewhere, some humoral influence of thymic origin, or some combina­ tion of these factors is involved. Gowans and his associates (1963) and Marchesi and Gowans (1964) discovered that these thymic dependent cells constantly recirculate from blood, into lymph nodes via venular wall transmigration, and back through the thoracic duct. Radio auto­ graphic studies using DNA-labeling with tritiated thymidine have shown that some of these small lymphocytes may survive the life span of the individual (Ford and Gowans, 1969). On stimulation with cer­ tain antigens, they may transform rapidly to become large blast cells with typical clusters of polyribosomes in their cytoplasm and the capacity to take up pyronin in histochemical staining. In vivo these activated cells are characteristically present in transplants, in direct contact with the foriegn cells of the graft at the time of early acute rejection (Porter, ej: ai, 1964a). These cells, more than any other type of cell are the transplant killer cells. The antibody 17 bound closely to their cell membrane probably imparts to them their

high degree of specificity (Biberfeld, al.. 1971) and (Greaves, et

al.. 1969). The nature of their destructive action against the cell

membranes of foreign cells is not established. It could involve a

portion of the complement system, particularly C 8, which is bound to

membranes (Perlmann, et al.. 1969). Activated lymphocytes have been

shown, however, to release a number of factors (macrophage inhibiting

factor, lymphotoxin, transfer factor, interferon, and probably factors

having to do with skin reactivity, chemotaxis and mitogenicity of

other cells). Information regarding effector substances has recently

been reviewed (Pick and Turk, 1972). A role for any of these mediator

substances in direct graft pathology has yet to be demonstrated.

The means by which host lymphocytes are sensitized against graft

cells is uncertain. Evidence has been advanced by Najarian and

associates (1966) that antigenic material can be recovered from the

renal-vein plasma within hours of kidney transplantation in dogs.

Sensitization of responsive cells could take place at a distance from

the graft if it could be confirmed that antigenic molecules circulate

free from cells. Strober and Gowans (1965), from their experiments

involving the perfusion of kidneys with labeled foreign thoracic-

duct lymphocytes, concluded that the cells themselves pick up anti­

genic stimuli peripherally. This could occur as they come into

contact with the endothelium of the transplant, or possibly during a

period of independent migratory activity outside vessel walls among

the cells of the transplant. 18

The arena of host response to foreign antigen is the lymphoid centers. As suggested above, the transport of antigen to these centers can be either in the form of free antigenic molecules, dis­ associated from their normal location as part of the structure of the cellular plasma membrane, or bound to recipient cells in some manner.

The route that these foriegn substances take in order to reach the lymphoid centers may be either via blood vascular channels or via lymphatic channels. Barker and associates (1969) have shown that a flap of skin, deprived of lymphatic drainage, but retaining a narrow vascular pedicle, can support the long-term survival of inlay skin allografts. If lymphatic connections are allowed to reestablish them­

selves, the foreign graft is rejected. Chanana and associates (1969) placed skin grafts over the posterior quarters of cattle and constant­

ly destroyed the thoracic duct lymphocytes by extracorporeal irradia­ tion. These grafts, the lymphatic drainage of which terminated in the thoracic duct, were preferentially protected from rejection. Other

lines of evidence that support the relative importance of lymphatic

over blood vascular channels as afferent conduits in the development of the immune response to sedentary tissue allografts are the findings of ‘'immunologically privileged” sites for transplantation. These

include, for example, the anterior chamber of the eye and the interior

of the brain. The survival of allogenic implants is supported in these spaces because they are devoid of lymphatic drainage in the

usual sense.

The possibility that both blood and lymph vessels are involved has not been excluded. It has been shown by White and Hildemann (1969) that first-set renal allografts usually outlive skin grafts between the same inbred strains of rats. This reflects on the quan­

titative significance of the lymphatic channels. Tilney and Gowans

(1971), however, have recently shown that rats used in their model do

eventually become sensitized to skin allografts on alymphatic pedicles

in a similar manner as renal allografts. These alymphatic pedicle

grafts contracted progressively from about 3 weeks after grafting. No

lymphatics could be demonstrated in the flap pedicle. Autografts on

similar pedicles remained unchanged. Second-set allografts applied

orthotopically were destroyed with a second-set tempo, indicating that

the first grafts had sensitized the host via the hematogenous route.

Once the antigenic message has reached appropriate lymphoid

centers, activated cells, the large pyroninophilic blast cells, appear

in considerable numbers. The dynamics of this phase of the response

to skin allografts have been described by Turk (1967). The changes in

the regional lymph nodes occur in an area called the paracortical area.

It is situated between the true cortex and the medulla where the small

lymphocytes are not so tightly packed as in the cortex. It lies

between and is distinct from the germinal centers of the lymph node

cortex. It can be shown that enlargement of this area can be indepen­

dent of any changes in germinal center activity in both contact sensi­

tivity and the allograft reaction. Enlargement of the paracortical

area of the draining lymph node is associated with proliferation of

lymphocytes. These paracortical areas have been shown by Parrott and

associates (1966) to be thymus-dependent in that they do not develop

in neonatally thymectomized mice. Lymphocytes differentiate into 20 large pyroninophilic cells which appear to divide into other lympho­ cytes in these areas of the lymph node.

Porter and associates (1964a) have studied in detail the role of the lymphocyte in the rejection of canine renal allografts. The cellular response of these dogs was investigated with tritiated- adenosine labeling combined with autoradiography and electron micro­ scopy. It was found that renal allografts caused large pyroninophilic cells to accumulate around the post-capillary venules of the lymph nodes and the splenic arterioles of the host. By 3 days many of the cells were in mitosis. Of the lymphocytes present in the thoracic duct lymph on the day of transplantation, very few entered the kidney; a larger proportion of the cells in the lymph at day 4 settled in the transplant. Some of the cells that appeared in the graft originated in the spleen after renal transplantation; none came from cells pre­ sent in the spleen before renal transplantation. The cells entered the graft by way of the peritubular capillaries. At 24 hours a few small lymphocytes were found in this capillary mesh work in the transplant, but from 48 hours onwards, most of the infiltrating cells had pyronin-positive cytoplasm and already resembled those in the lymph nodes and spleen. Some of these pryoninophilic cells had a well developed endoplasmic reticulum and appeared to be of the plasma cell series; others lacked this feature and resembled in many ways large lymphocytes or monocytes. Close association of the infiltrating pyroninophilic cells with the endothelial cells of the peritubular capillaries was followed by disintegration of the blood vessels.

Within the graft some of these cells underwent division, the total 21 cell cycle being about 12 hours.

The work of McCluskey and associates (1963) provided evidence for the specific attraction of sensitized cells to antigen but in addition provided several lines of evidence showing that the great majority of mononuclear cells which comprised the infiltrate in delayed hypersen­ sitivity reactions were not specifically sensitized cells. This information was provided by the experiments in which non-labeled cells from sensitized donors (guinea pigs) were transferred to non-immunized recipients which had been given tritiated thymidine in the 2 days pre- ceeding transfer. Delayed reactions elicited in these recipients were found to contain a high percentage (68 to 91$) of recently prolifer­ ated (labeled) cells.

The finding of severe vasculonecrotic lesions, without much accompanying cellular infiltration, in longer lived allografts

(Porter, et al.. 1964b), the demonstration of apparent antigen-anti- body complexes in the walls of vessels in kidneys which were under­ going rejection (Horowitz, et al.. 1965), the detection of cytotoxic antibodies in the recipient's serum after renal allografting

(Simonson and Altman, 1964), and the observations of hyperacute rejection of renal allografts in man (Williams, al.. 1968) support the role of humoral antibody in the allograft rejection. Early studies were carried out almost exclusively with grafts of skin, lymphoreticular tissue or serially transplanted tumors, small rodents for the most part being used as the experimental subjects. This work led to a widely accepted notion that, with the exception of disso­ ciated lymphoid and bone marrow cells, allografts were resistent to 2 2 humoral antibodies and that graft destruction was mediated by cell bound immune substances. However, more recent information provides clear evidence that both cellular and humoral responses are involved.

It has been shown that immune sera may have the power either to cur­ tail or prolong the expected survival times of grafts, depending on the source and quantity of the serum administered, the species and

strains of test animals and the kind of grafts employed.

The evidence weighs heavily in favor of the prime importance of

cell-bound antibody in the rejection of skin allografts, and the

strongest evidence in support of this is the repeated observation that passive transfer of amounts of antiserum far in excess of that

required for the destruction of grafts of lymphoid tissue or bone marrow fail to damage allografts of skin, except in the case of the white graft phenomenon (Stetson, 1959), whereas relatively small num­

bers of cells from immune donors are highly effective (Billingham, et

al.. 1954). Prolongation of survival times (enhancement) of skin

allografts after treatment of the hosts with antiserum specific for

antigens of the graft have been reported by Billingham, Brent and

Medawar (1956b) but the amount of prolongation was slight.

The involvement of serum antibody in the rejection of primarily

vascularized organs has been most clearly illustrated in renal trans­

plantation in man (Kissmeyer-Neilsen, e£ al.. 1966) and (Porter, 1967),

in which the presence, in recipients, of antibodies reactive with

donor tissues has been associated with prompt and generalized destruc­

tion of the grafted organ. There have been instances in which

antibody against blood group A and B antigens on renal vascular 23 endothelium have resulted in graft rejection (Gleason and Murray,

1967). The leukocyte antigens shared by most tissue are most often involved in an immune response. In these instances of hyperacute rejection, the circulating antibody has resulted from prior sensiti­ zation by transfusions or previous renal grafts. Williams and assoc­ iates (1969) have reported a hyperacute rejection in which the anti­ body was directed against a renal parenchyma cell antigen that could not be detected on leukocytes. These antibody reactions perhaps through the fixation of complement attract platelets and granulocytes, which have an important role in the development of an acute vasculitis that results in total or near total destruction of the transplanted organ (Najarian and Foker, 1969). Participation of humoral antibody

in late rejection of renal transplants in patients receiving immuno­

suppressive treatment is strongly indicated by studies that show a definite correlation between the development of vascular obliterative

lesions and the appearance of serum antibodies specific for donor antigens (Jeannet, e£ al.. 1970) and, the presence of immunoglobulins and complement in the obliterative lesions (Porter, et al.. 1968).

Renal allografts in dogs have also been found to be damaged by serum antibody, and again the lesions seem to affect the vasculature of the graft (Porter, 1964b).

There are several reports both from the use of dogs and rats that passively administered or actively acquired antibody against antigens of graft specificity have a protective effect on renal allografts

(Lucas, et al.. 1970) (Stuart, et al.. 1968) (1970) (Wilson, et al..

1969a and 1969b) and (Zimmerman, et al.. 1968). The mechanism of 24 enhancement or graft protection will be discussed in greater detail at a subsequent time.

The relative contribution of humoral and cellular mechanisms in

first-set (unsensitized) kidney graft rejection appears to depend upon the phylogenetic disparity of the donor and recipient species. Perper and Najarian (1966a) studied renal xenograft rejection in the widely disparate pig-to-dog experimental model. Whole organ xenograft rejection in these species was characterized by a hyperacute reaction

(10-20 minutes) manifested grossly and histologically by congestion, endothelial distruction and hemorrhage. The pattern of xenograft rejection was not altered by immunosuppressive drugs used singly or in combination (azathioprine, prednisone and actinomycin C). In this model the mechanism responsible for the immediate vascular rejection process involved preformed antibody, complement, vasospasm, clotting and sequestration of platelets and leukocytes. The relative roles of the effectors have been studied in an isolated perfusion system by

Mozes and associates (1971).

Recently Perper (1971) has described a pig-to-goat model in which, as judged by histological and immunofluorescent criteria, both cellular and humoral mechanisms participate in the rejection. The use of horse anti-goat lymphocyte globulin appeared to abrogate totally the cellular reaction, leaving a pure model of humorally mediated rejection. The average functional survival time was 3 days in untreated recipients and 11 days in those treated with antilympho­ cyte globulin.

The goat-to-sheep model, described by Perper and Najarian 25

(1966b), represented a condition in which cellular mechanisms pre­ dominate and humoral antibody can be demonstrated only as a partici­ pant in the rejection process. The histologic pattern of rejection was identical to allograft rejection (sheep-to-sheep) with non­ sensitized recipients rejecting xenografts slightly faster (11.3 days) than allografts (14.8 days).

The spectrum of immunological response to xenografts, from hyper­ acute to allogeneic-like, has been considered by Reemtsma (1971). He advances a scheme to explain differing responses in xenograft combina­ tions. Recipients of varying phenotypic patterns are exposed to cross­ reacting antigens. The role of Streptococcal and other heterologous antigens in transplantation has recently been reviewed by Rapaport

(1970a). It is quite possible that the acute rejection is a reflec­ tion of prior immunization of the recipient and that this immuniza­ tion is due to exposure to cross-reacting antigens.

In the allograft system, however, the variable which outweighs all others in its influence upon graft survival time is the genetic relationship between donor and recipient. The fact that genetical relationships were crucially important in transplantation was observed early in tumor research. The systematic reformulation of the entire theory of transplantation on genetically sound lines can be credited to C. C. Little (1923). Subsequent to Little, P. A. Gorer and G. D.

Snell made tremendous contributions in immunological genetics in their studies with the mouse. Mann and Fahey (1971) have recently reviewed histocompatibility antigens. Every species examined closely has been found to have many trans­ plantation antigen systems (ivanyi, 1970). At least one of these is

termed a major histocompatibility system, and it excites an immune

response which causes prompt and vigorous rejection of tissue grafts.

There are also minor histocompatibility systems which, individually,

cause less strenuous immune reactions, although when combined may

cause relatively rapid tissue rejection. In the mouse, for example,

there is one major histocompatibility system, termed H-2, and at

least 14 minor systems. Each histocompatibility system contains many

different antigenic specificities. These are grouped into related or

allelic series so that an individual member of a species has only a

few of the antigens. In man, for example, over 30 specificities are

known to occur in the major histocompatibility (HL-A) system but an

individual has only 2 to 4 of these specificities. The HL-A system

is governed by two subloci. There are 10 antigenic specificities

(alleles) coded for by the first sublocus and 20 by the second. One

allele codes for one antigen and there is, of course, only one allele

residing at each sublocus on each chromosome. The antigens coded for

by the alleles at each sublocus are spoken of as the first and second

segregating series. One chromosome will always code for 2 antigens.

Therefore, any individual will have no less than 2 major histocom­

patibility antigens nor more than 4. The genetic, chemical and

biological aspects of human histocompatibility antigens have recently

been reviewed (Amos, 1970a) (Reisfeld and Kahan, 1970) and (Walford,

Si aj,*, 1970).

Apart from the ABO system, the HL-A system appears to be almost 27 exclusively important in transplantation from a clinical point of view.

The other minor systems appear to be suitably controlled by immuno­ suppressions. Dausset (1971) has recently reviewed the role of the

HL-A system in transplantation.

Advances in knowledge of the histocompatibility systems of dogs have been a recent development (Epstein, ej: al.. 1968) (Cohen and

Kozaki, 1969) (Rapaport, et al.. 1970b) and (Dausset, et al.. 1971).

Using antisera useful for cytotoxicity typing and the one-way mixed leucocyte culture technique (Sullivan, et al.. 1971), a close corre­ lation between genetic relationship and survival of transplanted tissues has been shown just as in man. In a recent study of 268 sibling pairs from 11 litters, Templeton and Thomas (1971) obtained data that best fit a hypothesis of one locus on an autosomal pair of chromosomes with 20 alleles or more in the canine histocompatibility

(DL-A) system. The canine family studies of Dausset and associates

(1971) support this concept. The role of the DL-A system in trans­ plantation of kidneys (Rapaport, et al.. 1970b), hearts (Boyd, et al..

1970 and 1971) (Bos, et al.. 1971), lungs (Blumenstock, et al.. 1971), bone marrow (Epstein, et al.. 1971), liver (Chandler, et al.. 1970) and intestine (Westbroek, e£ al.. 1971) has recently been reported.

The role of the canine erythrocyte membrane antigens in trans­ plantation is not so clear. Naturally occurring alloantibodios against reciprocal erythrocyte antigens, as in the human AB system, have been demonstrated in the sera from 15 percent of 145 randomly chosen presumably untransfused dogs (Swisher and Young, 1961). There are no reports of any direct correlation between rejection of tissue 28 transplants and grafting across an erythrocyte antigen incompatibility or grafting in the presence of naturally occurring alloantibodies against the erythrocyte type of the graft donor. Altman (1965) demon­ strated that exposure of skin and renal allografts to a high titer of hemagglutinin directed against the donor does not cause acceleration of the rejection process. Only the A antigen and anti-A were studied in this report and the experiments were conducted on unmodified (not treated with immunosuppressive agents) recipients that rejected their grafts within 6 to 9 days. Histopathologic evaluation of rejected tissue presumably revealed only a "typical” cell-mediated allograft rejection.

As outlined above, it is clear that full immunologic responsive­ ness to the foreign antigens of a transplant can result in graft destruction. Chemical immunosuppression has been in large measure responsible for the level of success of clinical transplantation.

Azathioprine (Imuran) and corticosteroids, especially prednisone, have been valuable adjuncts in immunosuppressive treatment. Immuno­ suppression by chemical agents has been recently reviewed by Jean-

Francois Bach (1971). These chemical agents do not suppress immuno­ logic processes selectively, but interfere with synthetic processes and cell replication widely throughout the body as does whole-body irradiation. On the other hand, heterologous antilymphocyte globulin appears to preferentially inactivate a certain class of lymphoid cells.

Antilymphocyte globulin (ALG) recovered from an individual sensi­ tized with lymphoid cells derived from a foreign species have been 29 tested in biologic systems at least since Metchnikoff's studies in

1899. Waksman and associates (1961), Woodruff and Anderson (1963) and Guttmann and associates (1967) early studied the effects of such sera on graft survival. Several recent reviews have been devoted to the subject of heterologous antilymphocyte globulin (Mitchison, 1970)

(Medawar, 1969) (Sell, 1969) and (Perper, et al.. 1971).

Administration of heterologous ALG reduces the peripheral lympho­ cyte count to very low levels within hours. After chronic administra­ tion, histologic examination of lymph nodes shows that small lympho­ cytes are depleted from the paracortical areas, leaving outer parts of the nodes intact, and a similar depletion of cells takes place from the periarteriolar areas of the spleen -- both are "thymus-dependent" areas. The thymus itself is reduced in size but there is no obvious change in the normal architecture (Parrott, 1967). Lymphocytes attacked by ALG are removed from the circulation mainly in the liver

(Martin and Miller, 1967). Prolonged survival of several different experimental organ transplants when only ALG was used for immuno­

suppression has been achieved. Antilymphocyte globulin treatment is less effective against primary humoral responses and much less

influential in depressing secondary humoral responses than cellular

ones. The detection of humoral antibody in mice directed against the histocompatibility antigens of foreign skin grafts, still healthy and

in place on these mice being treated with ALG, is an example of this differential effect (Lance and Batchelor, 1968). A formulation has been recently presented by Lance (1970) on the preferential activity of ALG in depressing cell-mediated responses. Antilymphocyte globulin 30 binds to lymphocytes that are most accessible, these being principally those in the peripheral circulation. Approximately 7C$ of the lym­ phoid cells in the peripheral circulation are of the long-lived thymic-dependent type. Removal of these cells by direct, complement- dependent lysis or through the liver or spleen prevents their recir­ culation to the thymic-dependent areas in the lymph nodes and spleen and cell-mediated response is thus diminished. Short-lived bone- marrow-derived (plasma cell precursors) lymphocytes are rapidly restored but the thymus-dependent population may remain depleted for a long time. Thus, humoral antibody responses are relatively pre­ served. In fact, a humoral response against the heterologous protein

(ALG) leads eventually to a rapid immune elimination of the ALG thus diminishing its effectiveness in transplant protection (Amemiya, e£ al..

1970). Anaphylactic reactions to the heterologous globulin have also been reported.

To eliminate the complication imposed by antibody against a heterologous protein, several investigators have studied the use of a homologous antilymphocyte globulin. This antibody is produced in an individual by immunizing with tissue from a member of the same species but of a different allotype. Ono and associates (1969) reported on rat alloantisera that exhibited strain specificity and were immuno­ suppressive as demonstrated by the delayed rejection of allogeneic heart grafts in antiserum treated rats and the induction of lympho­ penia. Survival of skin allografts has been significantly prolonged in mice treated with anti-thymus alloantiserum (Taub, 1969).

So far all of the approaches to immunosuppression have been 31

"non-specific" in the sense that they suppress the body’s response to

a large number of different antigens at once. Specific suppression of

a host's capability to destroy a graft can be accomplished by applica­

tion of the enhancement principle and the induction of specific

tolerance to transplantation antigens.

Enhancement is an antibody-dependent immunologic phenomenon

resulting in prolonged survival or complete acceptance of an allo­

geneic graft. Certain experiments of Flexner and Jobling (1907) are

usually cited as the first examples of a favorable influence on the

growth of malignant tumor transplants between rats as a result of the

prior injection of heated tumor cell suspensions. Casey (1934)

established the specificity of enhancement in that the effect produced

by prior treatment with devitalized cells of a tumor led to enhance­

ment of that tumor only and not of another. Kaliss (1958) made the

important observation that the passive transfer of serum to mice

syngeneic with a treated animal, either before or after inoculation of

the appropriate tumor, could extend the capacity for supporting

enhanced growth of viable tumor cells. Furthermore, since it was

possible to produce enhancing antibodies by the injection of normal

donor-strain cells as well as tumor cells, it became clear that the

antigens concerned in these experiments were the allospecific antigens

of transplantation and were not in any way characteristic of tumors.

Recent reviews (Russell, 1971) (Amos, et al.. 1970b) and (Kaliss,

1970) are very informative of the current knowledge of enhancement and

its speculated mechanisms.

Immunological enhancement has been clearly shown to be the 3 2 mechanism of renal graft prolongation in the rat experiments performed by Stuart and associates (1968). Briefly, kidneys were transplanted across a major genetic barrier (Ag-B locus) from Lewis x BN hybrid rats into bilaterally nephrectomized Lewis rats. Grafts continued to survive with normal functions 72 to 142 days after grafting in rats treated singly and in combination with donor spleen cells administered intravenously one day before grafting and passive immunization with antidonor serum prepared in rats of the recipient strain. The host's immune response to other antigens was not impaired. Lucas and asso­ ciates (1970), using the same rat model reported indefinite renal graft survival in Lewis recipients pretreated with homologous anti­ lymphocyte sera (Lewis anti-BN). Such treatment did not prevent the rise in cytotoxic and hemagglutinating antibodies. Antibodies per­ sisted indefinitely in control animals in which the kidney was permitted to reject. Antibodies in antisera-treated animals, however, disappeared by the twenty-fourth day. Lymphocytes from Lewis rats bearing viable LBN kidneys were fully able to react with BN or LBN cells in mixed leukocyte culture. Appreciable amounts of antibody detectable by binding or binding-inhibition assays were present in all preparations of antilymphocyte serum having enhancing ability. These results suggest that "binding" antibody reacts with BN antigens in the transplanted kidney and protects it from humoral and cellular des­ truction by the host.

Recently Guttman and associates (1972) have reported on observa­ tions of prolonging renal graft survival in rats by the induction of enhancement by active immunization with a single intravenous injection 33 of cells from the thymus, spleen, lymph nodes, and whole blood when given under proper dosage and timing conditions. Barkin and associates

(1972) have seen prolongation of skin graft survival in mice when the skin donor was pretreated with high titer recipient antidonor serum.

Moreover, pretreated-skin graft survival was seen even in the pre­ sence of a simultaneous ongoing rejection response to an unpretreated- skin graft.

Prolongation of canine renal allograft survival has been reported by Wilson and associates (1969a) after pretreating with a donor specific solubilized spleen cell preparation and low doses of methyl prednisone and azathioprine. They explain the mean survival of 144 days for a group of 11 dogs that were treated as immunologic enhance­ ment. The route of administration, dose, source and timing of injec­ tion of the protein subcellular antigenic materials were of major importance in producing the desired effect. Similar results using the canine model have been reported (Zimmerman, et al.. 1963) and (Wilson, et al.. 1969b).

In a recent review by Russell (1971), active enhancement was considered as a double balance. The primary balance must favor humoral antibody production instead of cell-mediated immunity. The impact of any sensitized cells on the graft must be sufficiently restrained by humoral antibody directed against the same foreign specificities. Second, the effect on the graft of the humoral anti­ body produced must favor the survival of the graft by one mechanism or another instead of causing its destruction through direct cytotoxic activity. A widely accepted opinion in the field of immunological 34 tolerance to living cells is that highly tolerant animals build up no immunological reaction against the transplantation antigens of the tolerated line; and that the appearance of transplantation antibodies indicates the end of tolerance and leads to the destruction of the tissue previously well tolerated. Experiments by Voisin and Kinsky

(1968) presents results that possibly lead to a relationship between tolerance and enhancement. They injected mice at birth with allo­ geneic cells in order to be made tolerant; the tolerant state was evaluated by skin grafting. The results were as follows: l) As judged by studying the number of pyroninophilic cells and of immuno­ globulin producing cells, the lymphoid system of highly tolerant animals is in a state of immune reactivity greater than a non-immunized one. 2) Sera from highly tolerant animals very often contain low but detectable levels of antibody against the cells of the "tolerated" line as shown by direct hemagglutination or Coomb's test (indirect hemagglutination). The presence of these antibodies do not imply a breaking of tolerance (except for cytotoxic antibodies). 3) Sera from highly tolerant animals can contain specific enhancing anti­ bodies able to passively transfer to normal mice of the recipient

strain the ability to tolerate a tumor graft from the donor strain.

These antibodies can be specifically absorbed by cells having the corresponding H-2 antigens.

It is obvious from experiments such as those demonstrating graft

survival by mechanisms of enhancement or tolerance that the applica­ bility of organ grafting could be extended with superior results without the attending complications of complete and non-specific 35 immunosuppressive therapy. Immune tolerance and/or enhancement would provide for graft survival without crippling the protective mechanisms of the body which guard against infectious agents and neoplasia.

The Human Kidney Transplant Registry's eighth report (Murray, et al.. 1971) estimated that 87 percent of the renal grafts performed between identical twins after two years were still functioning. The longest survival was then 15 years having been performed in 1956.

Close blood relatives had an average two year survival of 75 percent; while kidneys from living unrelated individuals had a two year sur­ vival of only 66 percent. The survival rate of kidneys from unrelated cadaveric donors was 41 percent — the longest functioning cadaver kidney being 7 years. It is obvious that renal grafts from related

individuals are more successful than those from an unrelated indi­ vidual. It is also obvious that grafts from living donors have a

higher success rate than those of cadaveric origin. When using

living donors, time is available for histocompatibility testing and matching for optimal donor-recipient pairing, while the urgency

involved in using a cadaver's organ obten eliminates any possibility

for histocompatibility matching and pretreatment of the recipient with

immunosuppressive agents. Another important advantage in using a

living donor is the opportunity to minimize the ischemic damage that

often occurs in a cadaveric organ.

Despite factors in favor of living donors, there is concern on

the part of some in promoting their use because of possible distress

placed on family members. A study recently undertaken by Simmons and

colleagues (1971) has shown that the search for a kidney transplant 3 6 donor in a family can cause considerable stress and often leads to

strife within a family.

The current alternative, the use of cadaveric organs, could be made more successful if donor organs could be preserved for prolonged

periods. This would allow time for proper donor-recipient histocom­

patibility pairing. Irj vitro clinical preservation techniques now in

use consist of pulsatile perfusion and/or simple cooling and are con­

fined to relatively short periods of storage, 50 hours being the

maximum for a human kidney to date (Belzer and Kountz, 1970). Often

kidneys from cadavers have suffered ischemic damage and remain anuric

for weeks and months before function is resumed if ever. The ideal

organ preservation system would be one that identically matches the

normal physiological environment of the intact functioning animal or

man. Such a system might even provide for repair and recovery of a

damaged organ and an interim assessment of organ function prior to

definitive transplantation. With this type of in vivo environment,

prolonged storage could become feasible.

The original concept of .in vivo storage was developed by Acker-

mann and associates (Ackermann, ,§£ al.. 1966), who used an allogeneic

canine intermediate host model. One of the disadvantages in using an

in vivo system involves the immune response of the intermediate host

to the stored organ and the subsequent immune response of the defini­

tive host to intermediate host antigens adsorbed to the graft vascular

endothelium or to intermediate host cells within the parenchyma. In

order for the ,in vivo technique of storage to be applicable to man,

an intermediate host of a different species would be required. Sell 37 and Dupree (1966) coined the term "xenobanking" for such a procedure.

The model for studying xenobanking must consist of two different species but not so widely divergent that preformed antibodies mediate hyperacute rejection. Using a sub-human primate model of closely related species, Sell and Benjamin (1971) and Benjamin and Sell (1972) have modified intermediate hosts by total body lethal irradiation to prevent their immune response to the stored organ.

At the present time the most serious limiting factor in clinical transplantation is the supply of suitable donor organs. A means of organ preservation for prolonged storage would improve the problem.

The above mentioned technique of xenobanking is promising; and a model

suitable for this type of study would also serve as a model for xeno- grafting. The use of organs from a different but closely related

species would eliminate most ethical and legal problems now plaguing human transplantation. It would also diminish the problems of organ procurement if a suitable species could be raised for just such a purpose. The clinical experiences of Reemtsma and associates (1964b) with chimpanzee-to-human renal grafts and those of Starzl (1964) with baboon-to-man renal grafts point to the feasibility of clinical xeno-

grafting and justify continued studies in xenogeneic transplantation. EXPERIMENTAL PROCEDURE

Production of Rabbit Antibody Against Dog and Covote Serum

Rabbits were immunized with fresh serum from one dog or one coyote by an initial foot-pad inoculation with one milliliter (ml) of whole serum combined with an equal volume of complete Freund* s adjuvant (CFA, Difco Lab., Detroit, Michigan). Two subsequent sub­ cutaneous inoculations of one ml. of serum each were administered at ten day intervals. The antisera were harvested ten days after the third inoculation.

Interfacial Rina Precipitin Test

Dog and coyote serum was doubly diluted in 0.85E& saline and overlaid in 3 x 50 mm glass tubes with an equal volume (0.1 ml) of undiluted rabbit anti-dog serum or rabbit anti-coyote serum. These reactants were allowed to incubate at 2 2 ° C for 3 hours. The tests were interpreted as positive or negative at a given dilution by the presence or absence of a precipitate.

Absorption of Rabbit Antisera Against Dog or Covote Serum

Primary absorption was accomplished by incubating together at

4°C and at 2 2 ^ equal volumes of undiluted antiserum and a 1;4 saline- dilution of the appropriate antigen (either dog serum or coyote serum). The precipitate was removed from the absorbed antiserum by

38 39

centrifugation at 2300 g for 20 minutes. Three sequential absorptions were performed on each antiserum with both pooled coyote and pooled

dog sera. Each species' pooled sample included serum of the original

immunizing donor and sera from 4 other animals. A doubling dilution

of the antiserum resulted from each absorption. Antisera were used

in immunodiffusion tests at dilutions of 1:2, 1:4 and 1:8. Serum antigen was employed at a saline dilution of either 1:5 or 1:10.

Ouchterlonv Technique

The tests were performed on 3 x 1 in. glass micro slides. The

slides were first coated with an impregnation agar solution which was allowed to dry. This solution consisted of 0.1$ Difco Special Agar

Nobel and 0.05$ glycerine in distilled water. Next a uniform layer of

1$ buffered agar solution was poured on the slides to a level of approximately one millimeter (mm) thick. The use of LKB slide frames

facilitated the procedure. The buffered solution was prepared by dissolving one bottle of ready mixed Veronal (Barbital) buffer salts

LKB (3276-VB10) in distilled water to make 1,500 ml. of solution, pH

8.6, ionic strength 0.1. The 1$ buffered agar gel solution consisted of 1 part Difco Special Agar Noble, 25 parts buffered solution and 75 parts double-distilled water. Using an LKB gel punch, a center well and six peripherial wells two mm. in diameter were cut and the gel plugs aspirated. The center well was filled with antiserum and the peripherial wells filled with antigen prior to incubation at 2 2 ° C for

24 hours in a humid chamber. Following this incubation, the excess protein was washed off by allowing the slides to set immersed in a 1$ 40 sodium chloride solution for six hours. A second bath of fresh 1$ sodium chloride was used in a subsequent 16 hour immersion. Next the slides were immersed in distilled water for one hour then set out at room temperature to dry. When completely dry, the slides were immersed in 0 . $ amido black 10B in rinsing solution. The rinsing solution consisted of methyl alcohol, acetic acid and water in the proportion 45:10:45. Finally, the slides were rinsed free of excess dye in rinsing solution and dried at room temperature. Photographs were taken with a Cordis immunodiffusion camera (Miami, Florida) using Polaroid black and white land pack film type 107.

Immunoelectrophoresis Technique

Serum antigens and corresponding antisera were used undiluted in the immunoelectrophoresis technique. The glass slides and agar gel used were the same as those used in the Ouchterlony technique described previously. LKB immunoelectrophoresis apparatus was employed throughout this technique. Wells and troughs in the agar gel were cut to a width of one millimeter. Veronal buffer solution, prepared as described for the Ouchterlony technique, served as the electrolyte solution. Electrophoresis was performed for one hour at

250 volts and 50 milliamperes. Following electrophoresis, immune serum was applied to the trough and diffusion allowed to proceed for

20 hours at 2 2 ^ in a humid chamber. Washing, staining and rinsing of these slides was the same as that for the Ouchterlony technique previously described. 41

Horse Antibody Against Dog Cells (HADLG)

Lymphocyte suspensions were aseptically prepared from lymph nodes of dogs. The nodes were minced with scissors and pressed twice through a sixty gauge mesh sieve (W.S. Tyler Co., Mentor, Ohio) to achieve a suspension of separated cells. Chilled Hank's balanced salt solution (HBSS) with disodium dihydrogen ethylenediaminetetra- acetate dihydrate (EDTA) (G.F. Smith Chem. Co., Columbus, Ohio) added was used to facilitate filtering the cells through the sieve. The cell suspension was next washed twice with HBSS with EDTA using a speed of 400 g at 4°C in an International PR-6 centrifuge (Boston,

Mass.). Contaminating connective tissue debris and erythrocytes were separated from the mononuclear cells by isopycnic zonal sedimentation

(Perper, e£ al.. 1968) using Hypaque-Ficoll solution added at one- fourth the total volume of cell suspending medium. The gradient solution was prepared by adding 24 parts of % Ficoll (Pharmacia,

Piscataway, N.J.) in distilled water to 10 parts of 34^ Hypaque

(sodium Diatrizoate, Winthrop Lab., New York, N.Y.) in distilled water. Centrifugation at 400 g for 25 minutes at 2 2 ^ layered the mononuclear cells at the gradient-medium interface. The cells were collected by aspiration v/ith a pasteur pipette. Residual erythrocytes were lysed by resuspending the cells in distilled water for 30 seconds followed immediately with the addition of a concentrated solu­ tion of HBSS to regain isotonicity of the suspending medium. Follow­ ing a final wash, a total and differential cell count was made.

Lymphoid cells with less than one percent macrophage-type cell con­ tamination were used for inoculation. 42

A total of four inoculations of dog lymphocytes freshly prepared

each time were used to immunize the horse. The first inoculation con­

sisting of 2 x 10* cells in ten ml. of HBSS was administered sub- cutaneously in eight sites with an equal volume of CFA. Three subse­ quent intravenous inoculations were given at approximately three week

intervals using 17 x 10^, 15 x 10^ and 4 x 10^ cells respectively

suspended in 250 ml. of normal saline. The lymphocyte cytotoxic and hemagglutination titers were monitored periodically during the

immunization regimen. The immune serum was harvested eleven days after the last injection and stored until needed at -70°C after com­ plement inactivation at 56°C for one hour.

As soon as the immune horse serum was thawed, it was multiply absorbed with washed dog erythrocytes. Dogs were exsanguinated into

600 ml. sodium citrate containing bottles. These cells were washed five times with sterile saline using centrifugation speeds of 1500 g at 4°C in an International PR-6. After each centrifugation the

supernate and buffy coat were discarded. Volumes of packed erythro­ cytes prepared in this manner were used for the absorptions.

Absorptions of the immune horse serum were performed at 4 ^ with occasional stirring. The absorption periods were five hours to thirty hours long. The volume of packed cells used for a single absorption varied from one-fourth to one-half the volume of the antiserum. The volume of antiserum was generally 450 milliliters. After each absorp­ tion the cells were repacked at 1500 g for 25 minutes at 4 ^ and the antiserum removed and added to fresh erythrocytes. The agglutination activity for dog erythrocytes was monitored throughout the procedure 43 and absorptions discontinued when the hemagglutinating activity was removed or reduced to a very low level.

Eleven thousand five hundred ml. of the absorbed antiserum was then twice precipitated with 4C% saturated ammonium sulfate (J. T.

Baker Chem. Co., Phillipsburg, N. J.) in water. Briefly, the anti­ serum was precipitated in 100 to 125 ml. aliquotes. The volume of saturated ammonium sulfate to use for a precipitation with 40% ammonium sulfate was calculated as follows:

Ya = ys X 1 - X

Ya = volume of saturated ammonium sulfate

Ys = volume of antiserum aliquot

X = percent of ammonium sulfate desired (converted to a decimal)

The calculated volume of ammonium sulfate was then added drop- wise into the antiserum at 2 2 ° C with constant stirring over a period of 20 minutes. Following this addition, the mixture was stirred for two hours at 2 2°C to allow complete precipitation of the globulin.

Next the mixture was centrifuged in a Sorvall RC2-B (Norwalk, Conn.) for 25 minutes at 12,100 g at 2 2 ^ in 40 ml. tubes housed in an SS-34 head. The supernate was discarded and the precipitate dissolved in

0.9% sodium chloride and reconstituted to the original volume with

0.9% sodium chloride. Again, this material was precipitated as just described. After sedimenting the precipitate at 12,100 g at 2 2 ^ it was reconstituted to only one-third the original volume with 0.9% sodium chloride. This globulin material was then dialyzed against cold running tap water for 16 hours in cellophane dialysis bags (no 4 4 more than 75 ml. per bag). Following this, the material was dialyzed against 0 .% sodium chloride at 4°C for 24 hours. The sodium chloride bath was changed at least 7 times during this period. After this dialysis, the globulin solution was centrifuged at 17,300 g for 25 minutes at 4°C to remove small precipitates that may have formed. The supernatant globulin preparation was then collected, filter sterilized through a 0.22 micron millipore filter and frozen at -70*^3 in 10 ml. aliquotes.

The total protein of this product was determined by the Biuret method (Campbell, et al.. 1970). Electrophoretic analysis of the protein precipitate was performed on each batch. These were per­ formed on polyacetate strips (Sepraphore-3, Gelman, Ann Arbor, Mich.) at 300 volts and 30 milliamperes for 45 minutes. The strips were stained with Ponceau-S and analyzed on a recording electrophoresis densitometer (Densicord, Photovolt Corp., New York, N. Y.).

Production of Rabbit Antibody Against Dog and Covote Cells

Spleen cells were used in the immunization regimen for the rabbits. Spleen cells from a single coyote and from a single dog were used. Cell suspensions were prepared exactly as described in the preceeding section for lymph node cells. The first inoculation was with freshly prepared cells suspended in 2.5 ml. of HBSS while subsequent inoculations were with cells stored at - 7 0 ^ in medium ready for injection upon thawing.

The rabbits were immunized with a total of approximately 350 x

10^ cells administered intradermally and subcutaneously in three 45

inoculations at two week intervals. An equal volume of CFA was

included in the first inoculation. One week after the last injection,

the serum was harvested, heat inactivated and frozen at - 7 Q ° C .

Production of Dog and Covote Antibody Against Dog and Covote Cells

Immune sera were also obtained from dogs and a coyote after skin

graft rejection. The graft recipients were anesthetized and prepared

for an aseptic technique. Two full thickness grafts, three centi­

meters in diameter, were sutured in a prepared graft bed on the

lateral thorax of each recipient. Two dogs (3041 and 3052) were each

grafted with skin from two different dogs, 3049 and 3051 respectively;

a single dog, WPUP, received a graft from coyote 12; and coyote 12 was

grafted with skin from dog 3051.

The graft area was covered v/ith a sterile dressing postopera-

tively. After the third postoperative day the grafts were examined

daily until rejected. In some cases, a secondary inoculation of

approximately 150 x 10 viable spleen cells of graft donor origin prepared as previously described were administered intradermally in

order to achieve a lymphocyte cytotoxic titer greater than 1:256.

When each animal achieved a lymphocyte cytotoxic titer of 1:256

or greater, it was plasmaphoresed weekly, biweekly or every other week

in order to acquire a great quantity of the immune serum. The blood was collected from a jugular venipuncture into sodium citrated bottles. Two hundred to 600 ml. were taken at one time. The cells were packed by centrifugation at 1200 g for 25 minutes. The plasma was removed by aspiration and the cells resuspended to their original 4 6 volume with lactated Ringer's solution. These resuspended cells were then reinfused intravenously into the donor dog.

The plasma was clotted by adding 2 ml of calcium chloride for each 10 ml of 3.8^ sodium citrate present. The plasma was stirred while the calcium chloride was added, then incubated for 30 minutes at

3 7 ^ . After clotting, the serum was expressed by squeezing the cut up clots in a sterile gauze pack. Nine hundred to 1500 ml of serum was obtained from each dog in this manner. These antisera were then heated to inactivate complement and the globulin precipitated as des­ cribed previously for the HADLG. The sera precipitated included both dog anti-dog sera (DADLG) and the coyote anti-dog serum (CADLG). No absorptions were performed on this antiglobulin prior to ij} vivo use for immunosuppression.

Finally antisera were obtained from dogs sensitized with a coyote renal graft. Included in these grafting studies were dogs receiving no treatment, dogs treated with HADLG alone, dogs treated with cellular antigen alone, dogs receiving both HADLG and cellular antigen, and dogs receiving either DADLG or CADLG alone.

Dogs receiving HADLG alone were treated at a dosage level of 7 mg of gamma globulin per kg of body weight daily. This quantity of globulin for administration was selected as being higher than the average reported dose. Continued use of this dose was based on its capability to produce a profound lymphopenia without evidence of toxicity while prolonging graft survival in the first case performed.

Administration of 7 mg per kg was continued daily post-grafting until day 40 or until death if it came sooner. The product was administered 47

subcutaneously in multiple sites without evidence of producing pain at

the injection site. Transient slight edema was present after several

hours post-injection but abcessation was never observed.

The dosage level of DADLG (3041 anti-3049, 3052 anti-3051) and

CADLG (coyote 12 anti-3051) administered to graft recipients varied

from 7 to 14 mg of gamma globulin per kg body weight based on a dose-

response analysis of the antiglobulin in dogs whose cells in vitro

reacted maximally (1:256 or greater) with the antiglobulin. This

analysis was performed on DADLG only. An animal whose cells were of

the proper antigen type was not available to analyze the CADLG before

use on a graft recipient so a dose of 7 to 10 mg per kg of body weight was used.

Adverse effects of the globulin product on the host were also

considered in determining the dose and route (subcutaneous or intra­

venous) of administration. Graft recipients receiving DADLG and

CADLG were splenectomized at the time of grafting.

Spleen cells used as antigen were isolated as previously

described. The cells were then suspended to a concentration of 18 x

10^ cells per ml in HBSS and sonicated (Biosonik, Bronwell Scientific,

Rochester, N. Y.) for 60 minutes at 5 to 10dC at a maximum setting on

a 10 kilohertz per second set frequency. The preparation was examined

microscopically for complete cellular disruption. The solubilized

cell suspension was then centrifuged for two hours at 105,000 g at

5^C in a Beckman L2-65B ultra centrifuge (Type 40 fixed angle rotor)

(Pasadena, Calif.). The resultant supernate was frozen at -70°C. A

solubilized liver cell suspension of graft donor origin was similarly 4 8 prepared for use as antigen. The total protein content of each of those antigen preparations was determined by the Biuret method.

The solubilized antigen was administered intravenously to the dogs according to the following four dosage schedules.

1) A total of 10.5 mg of splenic antigen administered tri­

weekly for 13 injections ending on the day before grafting;

2) Same as (l), with the addition of HADLG administered sub-

cutaneously daily (7 mg per kg of dog body weight) starting

five days before grafting and continued daily post-grafting;

3) A total of 6.1 mg of splenic antigen administered tri-weekly

for eleven injections ending on the day before grafting.

HADLG administered as in (2);

4) A total of 23 mg of splenic antigen administered on the day

before grafting. Post-grafting administration of 23 mg of

liver antigen tri-weekly until death. HADLG administered as

in (2).

Renal grafts were placed in the recipient iliac fossa by anasto­ mosing the renal vein end-to-side to the common iliac vein and the renal artery end-to-end to the external iliac artery. The ureter was implanted into the recipient's bladder through a submucosal tunnel.

The recipient was bilaterally nephrectomized at the time of transplan­ tation.

Serum samples were taken from the dogs 3 to 5 times a week before and after grafting. Complete hemograms and blood urea nitrogen deter­ minations were performed 3 to 5 times a week. Water consumption, urine output, body temperature and general clinical condition was 49 recorded daily on each dog.

Cvtotoxic Assay

Antisera against cells were assayed by means of a micro cyto­ toxic test. Lymphocytes were isolated from peripheral blood either by Hypaque-Ficoll sedimentation as described previously or by Plasma- gel (Roger Bellon Lab., Neuilly, France) sedimentation. Using the latter technique, Plasma-gel was added in a volume that was one-third the volume of the blood and thoroughly mixed. The erythrocytes were allowed to settle and the leucokocyte-rich plasma removed before a buffy coat formed. This cell suspension was washed once with HBSS containing EDTA using a centrifugation speed of 400 g for 10 minutes. Contaminating erythrocytes were water lysed as previously described. Platelets, granulocytes, monocytes and fibrin debris were excluded from this cell suspension by a 15 minute incubation at 379c in a $ gum arabic-coated glass bead (0.2 mm diameter) column. The lymphocytes were eluted from this column by gravity using HBSS without

EDTA to flush them through. The lymphocytes were again washed using

slow speed centrifugation then resuspended in medium containing 30^ inactivated antologous serum at a concentration of 10 cells per milliliter.

One microliter of this cell preparation was added under oil to one microliter of doubling dilutions of antiserum in a microtiter plate (Falcon Lab., Los Angeles, Calif.) and incubated for 30 minutes at 37°C. Four microliters of rabbit complement was then added followed by a final 30 minute incubation at 3 7 ° C . 50

The same pooled source of complement stored at - 7 0 ^ was used for all cytotoxic tests performed. The addition of 5 microliters of 3%

Eosin-Y and 4 microliters of 1($ buffered formalin completed the test.

Cell viability was determined by inverted phase microscopy. Cell death greater than 2C^ constituted a positive at a given serum dilu­ tion providing negative controls (cells and medium; cells and comple­ ment and medium; cells and complement and normal serum and medium) contained less than 3% dead cells.

Preparation of Complement Source

The complement source was a pool of the sera from five rabbits.

Each rabbit was exsanguinated by cardiac puncture and the separate samples allowed to clot at 2 2 ° C for 30 minutes. Clot retraction was achieved by letting the clot set at 4°C for 4 hours. The serum was then harvested and immediately frozen at -70°C.

Using a single dog's cells and an antibody (HADLG) known to be cytotoxic for dog lymphocytes, a serum sample from each rabbit was analyzed for its complement activity and for the presence of naturally occurring anti-dog lymphocyte cytotoxin. All the sera contained the unwanted cytotoxin; so a sample of freshly thawed serum from each of the 5 rabbits was pooled and absorbed v/ith dog spleen cells to ascertain the most effective means of removing the cytotoxin v/hile leaving the complement activity at a usable level.

The spleen cells were prepared by mincing the tissue and pressing it through a 60 gauge sieve once. The cells were washed three times with HBSS prior to use. Absorption of the undiluted serum was per- 51 formed with an equal volume of spleen cells at 4^C. Each absorption period was 2 hours long. One sample was absorbed once; another sample was absorbed twice; while a third sample was absorbed three times. After the completion of each samples' absorption, it was immediately frozen at -70°C. An unabsorbed serum sample was held at

4°C concurrent with each of the three absorptions to serve as a con­ trol on complement activity decay while being held at 4 ^ for several hours.

The three differently absorbed complement samples were then used undiluted and at dilutions of 1:2, 1:4 and 1:8 in cytotoxic tests.

Based on optimal enhancement of cytotoxicity without causing cell death in control cultures, it was concluded that the undiluted sample having been absorbed one time was satisfactory as a source of comple­ ment. This complement source was then vialed in aliquotes of one ml or less and frozen at -70^.

Direct Hemagglutination Test

Hemagglutination titers were determined using the direct saline hemagglutination test. Doubling dilutions of antisera were made in test tubes 13 x 100 mm with 0 . 8 % sodium chloride. Erythrocytes were washed three times and used in the test in a % suspension in 0 . 8 % saline. Equal volumes (0.1 ml) of cells and serum were incubated together at 37°t for 30 minutes. A negative control of cells and saline was included each time. Test evaluation was made by observing the cells while gently rocking the tube until all the cells dislodged from the glass. Reactions were recorded as follows: 52

S = solid cell button floated into medium

4 = solid button broken into few particles

3 = many large particles detaching from button as tube

was gently rocked

2 - several large particles in suspension with a background

of many small particles and free cells

1 = many small particles in a medium with many free cells

to give a granular appearance

i = few very small particles (specks) in a medium of free

cells.

Indirect Hemagglutination Test

The indirect hemagglutination test (passive hemagglutination test) was performed according to the method outlined by Campbell and associates (1970). Sheep erythrocytes aged for three days in Alsever's solution at 4°C were tanned with a 0.005S& solution of tannic acid

(Merck and Co., Rahway, N. J.) in saline. The cell suspension was standardized to an optical density of 0.5 at 520 nanometers on a

Bausch and Lomb Spectronic 20 spectrophotometer. Normal horse globu­ lin at a concentration of 0.25 mg per ml in saline served as the coat­ ing solution. The normal horse globulin was prepared as previously described for the ammonium sulfate precipitation of horse antibody against dog cells. Serum from renal grafted dogs treated with HADLG was reacted with horse globulin coated cells and uncoated cells.

Negative controls consisted of normal pretreatment serum from each grafted dog reacted with coated and uncoated cells and coated and 53 uncoated cells in medium. The diluent medium contained heat-inacti­ vated normal rabbit serum, 1:100, by volume, in saline to prevent spontaneous agglutination of the cells.

Several two-fold dilutions of antiserum were made in test tubes

(13 x 100 mm) starting at a dilution of 1:1000. Cells and sera were incubated together at 22°C for 3 hours before examining the pattern formed by the cells at the bottom of each tube. Each tube was scored

+, 1 or - in accordance with the following definitions (Campbell, et al.. 1970).

+ = compact granular agglutination or diffuse film of agglu­

tinated cells covering the bottom of the tube; edges of film

either folded or somewhat ragged.

1 = narrow ring of cells surrounding a diffuse film of agglu­

tinated cells.

- = heavy ring of cells or discrete smooth button of cells in

center of tube.

Mixed Leukocyte Culture Reaction

Peripheral blood, collected in heparin, was the source of lympho­ cytes used in the one-way mixed leucocyte culture reaction (M.L.R.).

The mononuclear cells were isolated by Hypaque-Ficoll sedimentation as previously described. Contaminating erythrocytes were minimal and their presence was of no concern. The mononuclear-rich cell suspen­ sion was consistently made up of approximately 7Cp& lymphocytes while the remaining cells were predominately monocytes with an occasional granulocyte. 54

The cells were washed twice with medium using slow speed centri­

fugation (200 g) before being put into culture. The medium was

minimum essential medium Eagle for suspension cultures (MEM-S,

Microbiological Associates, Bethesda, Md.) with 1% L-Glutamine, 100

units of penicillin per ml and 100 micrograms of streptomycin per ml

of medium added. The addition of % inactivated autologous serum to

the wash medium lessened the incidence of cell clumping during washing.

Cells to be used in a stimulatory capacity were incubated at

2 2 ° C for 30 minutes with 25 micrograms of Mitomycin-C (Sigma Chem. Co.,

St. Louis, Mo.) per 50 x 10^* cells suspended in approximately 2 ml of medium. The cells were kept in suspension by an occasional swirling

to prevent their sedimenting and creating high acid conditions by

close contact. Mitomycin-C rendered the treated cells incapable of blastogenesis without altering their ability to be stimulatory to a

second cell population. Following treatment, the cells were washed

twice to remove excess Mitomycin-C.

All cell preparations contained greater than 95 percent viable

cells at the start of the culture period as determined by dye exclu­

sion using % Eosin-Y solution. The cells were cultured in 3 ml of medium in round bottom glass tubes (16 x 150 mm) with metal closures.

Twenty percent inactivated autologous serum was added to the medium

for culturing the cells. Responder cells were cultured at a concen- tration of 1 x 10° or 2 x 10 cells per culture. Stimulator cells were added in equal numbers to the responder cells or at twice their concentration. Cultures were set to incubate at 37°C in a 5^ CO2 55

atmosphere. Mixed cultures and their controls were incubated for 5

or 6 days, while cultures stimulated by phytohemagglutin (PHA, Difco

Lab., Detroit, Mich.) or Concanavalin-A (Con-A, Calbiochem, San Diego,

Calif.) were incubated for three days. Phytohemagglutin (0.002 ml)

or Con-A (40 micrograms) were added per culture at the beginning of

incubation.

Blastogenic transformation of the responder cells was evidence of

stimulation. The degree of stimulation was ascertained by measuring

the amount of tritiated thymidine (%-thymidine, Schwarz/Mann, Orange­

burg, N. Y.) incorporated by the blast cells. Tritiated thymidine, 2

microcuries, was added to each culture 20 hours before harvesting.

The ^H-thymidine was of 1.9 curies per millimole specific activity.

The cells were harvested and combined with a scintillation

cocktail for one minute counts in a Packard Tri-Carb Liquid Scintilla­

tion Spectrometer with a teletype printout. The harvesting procedure

was as follows. The culture tubes were centrifuged at 250 g for 5

minutes. After discarding the supernate, the sides of the tube were

washed once with 2 ml of saline and again the cells were pelleted by

centrifugation. After discarding the supernate, the cell pellet was

disrupted with 2 ml of cold % trichloroacetic acid (TCA) using a

vortex mixer. Again the cells were centrifuged and the supernate

discarded. The precipitate was then dissolved in 0.5 ml of 0.1 N

sodium hydroxide followed by reprecipitation with 2 ml of TCA.

After pelleting the precipitate and discarding the supernate, 0.5 ml

of hydroxide of hyamine (Packard, Downers Grove, 111.) was added and

the precipitate allowed to completely dissolve (matter of a few 5 6 minutes). The dissolved cellular suspension was then decanted into a plastic counting vial. The tube was rinsed once with 1 ml of absolute ethanol and three times with a total of 15 ml of Bray's scintillation fluid, all of which were added in the plastic counting vial with the dissolved cells. The scintillation counting fluid was prepared with scintillation grade reagents. One liter consisted of 60 grams of

Naphthalene (Packard, Downers Grove, 111.), 4 grams of 2,5-diphenyl- oxazole (Packard), 0.2 grams of 1,4-bio-[2-(5-phenyloxazalyl^j -Ben­ zene (Packard), 100 ml of methanol, 20 ml of ethylene glycol and

1,4-dioxane (Packard) to make one liter.

Immunofluorescence

The method of demonstrating cell surface antigens by immuno­ fluorescence was essentially that described by Moller (1964) for viable cells using the indirect technique. Peripheral blood was collected in heparin. The mononuclear cells were separated from the erythrocytes by Hypaque-Ficoll sedimentation as previously described.

The erythrocytes were washed three times and resuspended to a concen­ tration of 20 x 10^ cells per ml in MEM-S. The lymphocytes were

isolated by incubating the mononuclear cell suspension in a glass bead column as previously described. Contaminating erythrocytes were excluded by water lysis as previously described or by lysis with

0 . 3 $ sodium chloride which was less harsh on the lymphocytes. The cells contaminated with erythrocytes were suspended in 10 to 20 times their volume with 0.3!$ NaCl and allowed to incubate at 22°C for 5 minutes. The lymphocytes were then pelleted at 200 g, washed twice 57 with medium then resuspended to a concentration of 20 x 10^ cells per milliliter.

The allogeneic antisera monospecific for a given canine blood group antigen were kindly supplyed by Dr. Robert Bull of Michigan

State University. Antisera against blood group antigens Aj_, C and D were tested. The antiglobulin used was rabbit anti-canine gamma globulin conjugated with fluoresce in isothiocyanate (FITC, Miles

Laboratories, Kankakee, 111.). Rhodamine conjugated bovine albumin

(Microbiological Assoc., Bethesda, Md.) in some instances was added to the fluoresce in conjugated material for contrast. Dilutions of alloantiserum used in the test were 1:2, 1:8 or 1:16 in saline. The conjugated material was used at a dilution of 1:5. When Rhodamine was added to the fluoresce in conjugate, it was prepared as follows.

The FITC was diluted 1:4 in saline. One part Rhodamine conjugate was added to four parts FITC conjugate to make a final dilution of FITC

1:5 and Rhodamine 1:5 in the conjugate preparation.

The test and controls included cells and antiserum and conjugate, cells and normal serum and conjugate, and cells and conjugate. Two million cells (0.1 ml) were added to a small conical centrifuge tube and centrifuged at 150 g for 2 minutes. The supernatant medium was removed and replaced with 0.1 ml of anti-serum or normal serum. The cells were resuspended and incubated for 15 minutes at 22^. After incubation the cells were washed 3 times in 0.2 ml of medium each time using the slow centrifugation speed for 2 minutes. After the last washing 0.1 ml of the conjugate was added and the cells brought back into suspension. They were incubated for another 15 minutes and 5 8 washed twice as described above. The cells were resuspended to their original volume in medium and inspected in a Zeiss fluorescent micro­

scope. Photographs were taken with a Nikon camera using Kodak Tri-X

Pan black and white film (TX 135) ASA 400. The film was developed at twice the ASA rating.

Canine Ervthrocvte Typing

Monospecific allogeneic typing sera for antigens A^, A 2, B, C and D were available for typing all dogs throughout this study.

Antisera specific for antigens F, Tr and He were added to the typing panel in more recent typings. The tests were performed in small test tubes using antisera having titers of 1:16 to 1:32 which v/ould cause macroscopic agglutination. The red cells being tested were prepared as a 4% suspension in isotonic saline. The use of fresh serum (com­ plement) enhanced the agglutination reaction of A^-anti-A^ as well as providing for a hemolytic reaction. Equal parts, 0.1 ml, of the antiserum and of the red cell suspension were mixed and allowed to

stand for 15 minutes at 3 7 ° C and then centrifuged for one minute at

1000 RPM. Those cells not reacting with anti-A^ and showing no macro- or microscopic reaction with anti-A2 were then subjected to the antiglobulin test using anti-canine globulin to serify the presence or absence of the A2 antigen.

Histooatholocrv

The biopsy samples were fixed in 1C$ buffered formalin. After dehydration and clearing, the tissues were double embedded in. paraffin.

The paraffin blocks were sectioned at 5 micra. Staining was accomplished with hematoxylin and eosin. RESULTS

Rabbit Antibody Against Dog and Covote Serum

The day following the third, and final, subcutaneous inoculation of dog and coyote serum into respective rabbits, necrosing arthus- type lesions were evident. A serum sample from each rabbit was collected six days later for antibody evaluation. The anti-coyote and anti-dog sera (RACS and RADS) were positive in dilutions of 1:1,024 and 1:32,000 respectively when reacted with homologous serum in the interfacial ring precipitin test. The greater quantity of antibody in the RADS became more evident during the absorption procedures. A heavy precipitate formed within 60 minutes of mixing each antiserum with diluted homologous or heterologous serum while being incubated at

22°C. Following the second absorption at for 18 hours, a preci­ pitate was visible in the mixture of RADS with either homologous or heterologous serum, but none was visible in the RACS mixed with homo­ logous or heterologous sera. Neither serum retained activity follow­ ing a third absorption first at 2 2 ° C for 2 hours and then at 4^C for

2 hours using the same mixtures as before.

Plate 1 depicts the reactions obtained following the incubation of RACS with dog and coyote sera in an Ouchterlony test. There were varying numbers and densities of precipitate but no differential 61

Plate 1. Rabbit anti-coyote serum (AC) reacted with dog (D) and coyote (C) sera in the Ouchterlony Test. (AC) = 1:2 dilution; (D) and (C) = 1:10 dilution Plate 2. Rabbit anti-dog serum (AD) reacted with dog (d ) and coyote (C) sera in the Ouchterlony test. (AC) =1:2 dilution; (D) and (C) = 1:10 dilution 63

Plate 3. Rabbit anti-coyote serum (AC) reacted with 6 different coyote (C) sera in the Ouchterlony Test. (AC) = 1:2 dilution; (C) = 1:10 dilution 64

Plate 4. Rabbit anti-dog serum (AD) reacted with 6 different dog (D) sera in the Ouchterlony Test. (AC) = 1:2 dilution; (D) = 1:10 dilution 65 indication of species of origin of the serum samples. Likewise, RADS depicted in Plate 2, demonstrated several precipitating systems with both dog and coyote sera but none unique to the dog. Plate 3 illus­ trates that the reactivity of RACS was not the same against every coyote serum tested. The same was true with RADS reacted with dog sera (Plate 4).

Plate 5 represents the manner in which antibody activity for dog and coyote serum (a) was removed from the RADS by absorption with dog serum. One absorption (b) diminished antibody activity for both species to a faint precipitate and a second absorption removed all antibody for both dog and coyote serum. While one absorption of RADS with coyote serum (c) left approximately one-half the precipitate density, no line of precipitate unique to the dog was retained. A second absorption removed all precipitating activity for both species.

Complete removal of antibody activity from RACS for both species

(Plate l) could be accomplished by one absorption with coyote or dog serum.

The immunoelectrophoretic patterns of RACS with coyote (bottom) and dog (top) sera is shown in Plate 6. Plate 7 represents the immunoelectrophoretic pattern of RADS with a dog serum (top) and a coyote serum (bottom). Each anti-serum reacted against both sera, regardless of whether from dog or coyote in almost an identical fashion. There was one difference in precipitate patterns but it was not species differentiating. Both RACS and RADS, when reacted with the serum from dog 123, failed to produce a line in the alpha 2 region (arrow) that was clearly visable when the two antisera were Plate 5

a. Rabbit anti-dog serum (AD), unabsorbed (U), reacted with the sera of 2 different dogs (D1 and D2) and 2 different coyotes (Cl and C2) in the Ouchterlony Test. (AD) = 1 : 2 dilution; (D) and (C) = 1:10 dilution.

b. Rabbit anti-dog serum, absorbed one time with pooled dog sera (APD), reacted with the sera of dogs and coyotes (same as in a) in the Ouchterlony Test.

c. Rabbit anti-dog serum, absorbed one time with pooled coyote sera (APC), reacted with the sera of dogs and coyotes (same as in a) in the Ouchterlony Test.

66 67

Plate 6. Immunoelectrophoresis of dog (D) and coyote (C) sera reacted v/ith rabbit anti-coyote serum (AC). Serum antigens and antiserum used undiluted. (+) anode; <♦> precipitinogen in the alpha-2 region (see text) 68

Plate 7. Immunoelectrophoresis of dog (D) and coyote (C) sera reacted with rabbit anti-dog serum (AD). Serum antigens and antiserum used undiluted. (+) anode; (!) precipitinogen in the alpha-2 region (see text) 69 reacted with coyote serum. It was assumed that both the dog donor and the coyote donor of the sera used for immunization had an antigen recognized by the responding rabbits and lacked by dog 123.

Horse Antibody Against Poo Cells (HADLG)

Figure 1 illustrates the manner in which antibody against dog lymphocytes and erythrocytes increased during the immunization proce­ dure. The horse used here had no preformed cytotoxic activity for dog lymphocytes; however, a naturally occuring hemagglutinin titer of 1:16 was demonstratable. Eleven days following the fourth inocu­ lation, the serum was harvested by carotid artery exsanguination. The fresh serum at that time exhibited a lymphocyte cytotoxic titer of

1:512 and a hemagglutination titer of 1:524,288.

Absorption of the immune serum with washed dog erythrocytes prior to ammonium sulfate precipitation effectively removed or markedly diminished the hemagglutinin activity. Figure 2 represents the absorptions performed on horse anti-dog lymphocyte serum (HADLS) batch lb. These results represent the absorptions performed on all batches. The cytotoxic titer was unaltered by the absorptions.

Following ammonium sulfate precipitation of each batch, the globulin product reacted with titers of 1:512 to 1:8,192 against dog lympho­ cytes. Table 1 depicts the globulin product's characteristics. These three are representative of the twenty-five batches precipitated.

A sample of the HADLS frozen at the time of exsanguination was thawed several months later and reacted with dog and coyote cells.

The reactivity of the antiserum with erythrocytes from both species Reciprocal of Serum Dilution ,524288— Figure 1. Horse anti-dog lymphocyte serum: Progression of antibody of Progression serum:lymphocyte anti-dog Horse 1. Figure 131072- 131072- 262144 - 32768 - 65536: — 16384 - •4096 — .2048 - 512 - 2 9 1 8 128 256 1024 “ 32 1 ie ih immunization. with titer 34 3 2 5 / / weeks / / / 6 / / \ / A. 8 7 Hemagglutination Titer Hemagglutination Titer cytotoxic Lymphocyte Exsanguination • Exsanguination Inoculations \ / \ V 1 1 12 11 10 9 /

70 I

71

524288 1

262144 \ Lymphocyte Cytotoxic Titer 1310721 - \ Hemagglutination Titer 65536 - \ \ 32768 - \ 16384 - \

\• 8192“ \ i \ 4096; - • \ ) \ 2048;- \ c • o •H \ -PD 1024 > H •H \ Q 512 — £ D Q>Jh CO 256 O 128 m \ o o \ h cu 64 ' *H \ O

8

4

2

0 I I I I I I 2 ' 3 4 5 6 7 No.‘Absorptions 3.5 17 25 15 7 13 19 Hours per Absorption

Figure 2. Horse anti-dog lymphocyte serum: Serum absorption v/ith dog erythrocytes. 7 2 is shown in Table 2. The hemagglutination titer varied from 1:2048 to

1:8192. Before frozen storage, this antiserum had a hemagglutination titer of 1:524,288. Some activity was apparently lost during storage; however, the strength of reactivity at each positive dilution was identical for both the fresh and frozen samples of antiserum. Both the normal serum and the immune serum reacted with dog and coyote cells in a non-species differentiating manner.

No distinction between dog lymphocytes and coyote lymphocytes could be demonstrated with the cytotoxic test. The reactions obtained were of the same order of magnitude, regardless of the cell source.

Table 3 illustrates these comparable reactions. Five absorptions of undiluted antiserum with lymphoid tissue from either species resulted in only a slight (one to two dilution) reduction of cytotoxic activity without any preferential absorption being seen. A single absorption of the antiserum at a dilution of 1:64 with either dog or coyote tissue completely removed all activity for the cells of both species.

Rabbit Antibody Against Dog And Covote Cells

Table 4 shows that while rabbit antibody reacted at different levels against the cells of individual animals of both types, absorp­ tions with dog tissue or absorptions with coyote tissue removed all activity for both homologous and heterologous cells. Thus, again,

species dependent differences were not demonstratable.

Dog and Covote Antibody Against Dog And Covote Cells: Skin Grafting

The allogeneic dog skin grafts sutured onto the two dog - recipients (3041 and 3052) were less than 1Q& viable on the sixth day TABLE 1

HORSE ANTIBODY AGAINST DOG LYMPHOCYTES

CHARACTERISTICS OF THE AMMONIUM SULFATE PRECIPITATED PRODUCT

Globulin Product lb Ic Id

No. RBC Absorptions (hr) 7 (99k) 7 (93) 6 (80|r)

Cytotoxic Titer 1:2048 1:512 1:8192

Hemagglution Titer 0 0 1:4

Total Protein 1.9 gm/lOO ml 3.1 gm/lOO ml 2.6 gm/lOO ml

Albumin 1.4% = .027 gn% 3.8% = .1 gn% GLOBULINS: Alpha 2 11% = .285 gn%

Beta 1 7.1% = .136 gn% % - .24 gn%

Beta 2 32.= .625 gn% 34.5% = 1.06 gn% V& = .44 gn%

Gamma 58.5% =1.11 gn% 65.5% = 2. gn% 57% = 1.48 gn%

73 TABLE 2

REACTIVITY3 OF HORSE ANTI-DOG LYMPHOCYTE SERUM

IN THE DIRECT HEMAGGLUTINATION ASSAY

Cell Source

Titer*3 Coyote 3 Coyote 5 Coyote 6 Coyote 7 Dog 3045 Dog 3047 Dog 3039

4 SSS SS SS 8 S S 4 S 4 4 S 16 4 4 4 4 4 4 S 32 4 4 4 4 4 4 4 64 4 4 4 4 4 3 4 128 4 4 3 3 3 3 4 256 3 3 2 2 3 2 4 512 2 2 1 1 2 2 4 1024 1 1 1 1 1 1 3 2048 1 1 1 1 1 1 2 4096 — — — — — 1 8192 1

Reactivity of Normal Horse Serum

4 2 1 1 1 1 1 2 8 1 1 1 1 — 1 1 16 1 1 _ — 1 1 32 — — 1 —

3 S = Solid; 4, 3, 2, 1 = Strength of Agglutination k Expressed as reciprocal of Serum Dilution 75 post-grafting. The dog skin placed on the coyote was less than 1Q& viable on the eleventh post-operative day; while the coyote grafts on the dog recipient were not considered rejected until the thirteenth day after grafting. This latter recipient was only eight weeks old.

Figures 3 and 4 illustrate the immune response of the recipients to skin grafting and lymphoid cell inoculation. No cytotoxic antibody was demonstratable in the serum of the dog immunized with coyote skin

(Figure 3) when reacted with donor cells until after a booster injec­ tion of 150 x 10^ skin donor lymphocytes. This was possibly indicative of the recipient’s lack of immunologic maturity.

Serum samples from the animals represented in Figure 3 were collected on the seventy-seventh post-graft day for use in subsequent cytotoxic tests. Serum from the animal represented in Figure 4 was collected on the seventeenth post-graft day for use in cytotoxicity testing. Serum samples were harvested from each animal at the inter­ vals indicated in Figures 3 and 4. The serum samples taken from an individual animal were pooled prior to ammonium sulfate precipitation.

Table 5 depicts the characteristics of the precipitated globulin products of the three immune serum pools.

The ability of these globulin products to effect a lymphopenia in dogs whose cells reacted in vitro to the same magnitude as cells from the immunizing donor was evaluated. This was done in order to obtain an indication of reasonable levels of these materials to be used in subsequent attempts at immunosuppression. Only 3041 anti-3049

ALG and 3052 anti-3051 ALG were evaluated. Figures 5 and 6 depict the leukocyte response of the host to the injections. The homologous 76

TABLE 3

HORSE ANTISERUM AGAINST DOG LYMPHOCYTES

Number of Animals 1 8 7 8

Coyote Dogs Dogs Dogs

Titer3 512 256 512 1024

Reciprocal of highest serum dilution showing greater than 2C$

cell death in the lymphocyte cytotoxic assay. TABLE 4

RABBIT ANTISERA AGAINST DOG AND COYOTE LYMPHOCYTES

UNABSORBED ANTI-DOG CELL SERUM UNABSORBED ANTI-COYOTE CELL SERUM Coyote Dog Dog Dog Dog Dog Dog Coyote Dog Dog Dog Dog Dog Dog 12 3049 3041 3051 3052 T OP-7 12 3049 3041 3051 3052 T OP-7 16 64 64 128 16 32 256 ' 256 64 32 128 8 512 512

Titer3

ABSORBED WITH DOG SPLEENb ABSORBED WITH DOG SPLEENb

0 0 0 0 0 0 0 0 0 0

------. ABSORBED WITH COYOIE SPLEEN0 ABSORBED WITH COYOTE SPLEEN*1

0 0 0 0 0 0 0 0

Reciprocal of highest serum dilution showing greater than 2C% cell death in the lymphocyte cytotoxic assay. bTiter checked only after the 5th absorption. cTiter after 2nd absorption. ^Titer after 3rd absorption.

77 78 globulin products produced a transient lymphopenia persisting only a matter of hours at dosage levels of 7 to 14 mg per kg and with some accompanying toxicity.

A lymphopenia was not observed after the first injection of 3052

anti-3051 ALG (Figure 5). It may have occurred without being detected

in the 8 hours before the first cell response evaluation was made. An

absolute decrease in the neutrophil count accompanied the lymphopenia after the second injection. Later injections provoked a neutrophilic

leukocytosis, giving evidence of inflammation and/or severe stress accompanying the administration of the globulin. Figure 6 represents

the peripheral cell response to the administration of 3041 anti-3049

ALG. A transient lymphopenia was observed simultaneously with a

neutrophilic leukocytosis.

The symptoms of toxicity manifested by the animals when injected

subcutaneously were apparent severe pain (yelping), muscular twitching and subsequent depression. Intravenous administration alleviated the

signs of pain somewhat but nausea, discomfort and depression remained evident. The severity of these "toxic" manifestations was in direct

proportion to the dose administered. A rabbit (3 kg body weight)

injected intravenously with 40 mg (13.3 mg per kg) and subcutaneously with 10 mg of the globulin product exhibited no adverse effects of the

treatment. The pH of the globulin products ranged from 6.8 to 7.

The antisera generated by skin grafting were tested against a

panel containing cells from 34 to 55 dogs and one available coyote.

The range of reactivity obtained is depicted in Table 6. The coyote cell reactivity is indicated by the double asterisk and does not Reciprocal of Serum Dilution 16&84 06 : 4096 “ 12 - 8192 04 - 1024 2048 1 - 512 5 ~ 256 32 \ Figure 3. Progression of Lymphocyte Cytotoxic of Lymphocyte Progression 3. Figure \ Days after skin grafting skin after Days

— — — — Coyote 12 response against dog 3051 dog against 12 response Coyote — — — — ..... £ Lymphoid Cell Booster Injection Booster Cell Lymphoid Plasma harvest £ | nioyPouto ih Immunization. with Production Antibody Dog VJPUP Dog 12 against Coyote response Dog 3052 response against dog 3051 dog against response 3052 Dog 4 1061294 81 70 20 79 Reciprocal of Serum Dilution 16384 4096 4096 “ 2048 - 1024 “ 512 512 “ 128 “ Figure 4. Progression of Lymphocyte Cytotoxic Antibody Antibody of Lymphocyte Cytotoxic Progression 4.Figure t * t i f rdcinwt Immunization. with Production 8 38 28 Dog 3041 response against dog3049 against 3041 response Dog injection booster cell Lymphoid Plasma Harvest Plasma 54 80 xlO cells per Figure 5. Dog (75) Dog Leukocyte toResponse 3052 anti-3051 5.Figure Antilymphocyte Globulin (ALG)Antilymphocyte Globulin administration. 14 mg/kg Hours • , , f • , 1514days mg/kg 7 0 . ~ N V — — -— - Seg. Neutrophils Seg. - -— — — TotalLeukocyte — — — ^ ALG ALG Administration ^ 28 I Lymphocytes xlO cells per. cmm 3 - 13 - 14 “ 15 2 1 / " i H 0j / , / 10 j- i" 8 :4 ; / — Sg Neutrophils Seg. — / 5 6;- !~ 7 9 j~ - 3 ‘“0 2 - > 1 iue6 Dg (99)toanti-3049 Response3041Leukocyte Dog 6.Figure i — — i , / - — = t = 123 456 78 7 6 5 4 3 2 1 0 / TotalLeukocyte “ ” / / / / niypoyeGoui (ALG) administration. Globulin Antilymphocyte Hours | ALG Administration ALG | ______Lymphocytes 2 8 83 appear to be different from that seen for the dog cells tested. In one instance it is non-reactive with a dog anti-dog cell serum and reacts at an intermediate level with a second dog anti-dog cell serum.

These unabsorbed sera behaved exactly as allogeneic antisera would be expected to. Four absorptions performed on each of these antisera with erythrocytes from dogs whose lymphocytes were reactive with the antisera did not remove lymphocyte cytotoxic reactivity noticably.

Table 7 represents the results obtained when dog antibody against coyote cells was absorbed with dog spleen. Although 4 absorptions always removed all activity for the absorbing cell and reactivity for certain other dog's cells was abolished or sharply diminished, in no instance did dog tissue remove all activity for coyote cells from this anti-coyote cell serum. On the other hand, when dog anti-coyote lymphocyte serum was absorbed with donor-specific cells, as shown in

Table 8, a similarity between the cells of the two species was shown since antibody activity against dog cells was removed or greatly diminished. The dog cells, in this example, that exhibit the least amount of antigenic similarity to the coyote cells in the cytotoxic assay are those most easily made non-reactors by absorption with specific coyote cells. In contrast, antibody activity for the dog cells that react most like those of the specific donor coyote is more difficult to absorb. However, as evidenced by the differential titers after 5 absorptions, there appears to be a close antigenic relationship between these dogs and the donor coyote.

Absorption of coyote anti-dog cell serum with dog spleen.(Table 9) again removed absorbing cell activity and removed or markedly reduced 84

TABLE 5

DOG AND COYOTE ANTIBODY AGAINST DOG AND COYOTE LYMPHOCYTES

CHARACTERISTICS OF THE AMMONIUM SULFATE PRECIPITATED PRODUCT

Coyote 12 Globulin Product 3041 anti-3049 3052 anti-3051 anti-3051

Cytotoxic Titer 1:256 1:256 1:128

Total Protein 1.55 gm/lOO ml 2.4 gm/lOO ml 1.8 gm/lOO ml

Albumin 0 3»5fo — .08 gn$ 0 Globulins Aloha 1 4. % = .062 onf', 30. % = .73 cats', 13.7^ = .25

Alpha 2 3 . % = .05 gn$ 9. % = .16 gr$

Alpha 3 8 • T/o — . 21 grfo 13.= .24 gr$

Beta 1 14 ./o = .22 grr?o 11. % = .26 grt% 31.8 % = .57 gn$

Beta 2 16.% = .24 gnfo 16. % = .38 gnfo

Gamma 6 2 vo = .98 gnfo 31. % = .74 gnfo 32.% = .58 gr$ TABLE 6

REACTIVITY OF DOG AND COYOIE ANTISERA AGAINST DOG A I'D COYOTE LYMPHOCYTES

RECORDED AS NUMBER OF ANIMALS REACTING WITH EACH ANTISERUM DILUTION

I'D TOTAL TITER3 REACTION 1 2 A 8 16 32 64 128 256 512 1024 ANIMALS

Dog 3041 Anti 21° 3 3 1 2 11 4 4 1 4b 2 56 Dog 3049

Dog 3052 Anti 1 1 2 4C 9 9 6 3b 1 36 Dog 3051

Doq W Pup Anti 8 1 2 3 . 3 4 1 2 5 4 2C 35 Coyote 12

Coyote 12 Anti 2 3 18 9 4b 36 Dog 3051

Reciprocal of highest serum dilution showing greater than 2C$ cell death in the lymphocyte cytotoxic assay. ^Donor of sensitizing cells. cCoyote included.

85 •TABLE 7

DOG W PUP ANTISERUM AGAINST COYOTE 12 LYMPHOCYTES

REACTIVITY OF ANTISERUM AFTER 4 ABSORPTIONS15

ADSORB i:-X3 CELLS REACTIVITY3 OF CELLS WITH D-l D-2 D-4 D-5 D-6 D-7 D-8 D-10 D-ll D-l 2 UNABSORBED ANTISERUM D-3 D-9

Coyote 12 1024 128 16 16 64 16 64 8 256 64 16 123 123

3051 16 0 0 0 0 0 0 0 4 2 0 0 0

3049 32 1 0 0 0 2 0 0 2 4 0 2 2

D-l 0 0

D-2 512 0

D-3 8 0

D-4 16 0

D-5 0 0

D-6 512 0

D-7 256 0

D-3 0 0

D-9 256 0

D-10 512 0

D-ll 32 0

D-l 2 128 0

Reciprocal of highest serum dilution showing greater than 2C$ cell death in the lymphocyte cytotoxic assay. “Absorptions performed with dog spleen cells. TABLE 8

DOG W PUP ANTISERUM AGAINST COYOTE 12 LYMPHOCYTES

REACTIVITY OF SERUM AFTER REACTIVITY OF ABSORPTION V/ITH COYOTE 12 SPLEEN ANIMAL UNABSORBED SERUM 2X ax 4X 5X

Coyote 12 1024 256 128 128 32

Dog 07 1024 64 64 16 4

Dog 3051 16 16 0

Reciprocal of highest serum dilution showing greater than 2Q& cell death in the lymphocyte cytotoxic assay. 88 activity for a second dog's cell; however, antibody activity against cells from the immunizing donor was not ever completely removed.

Table 10 shows that when dog anti-dog cell serum was examined, before and after absorptions with dog spleens, absorbing cell activity was always removed and activity for other reactive cells was removed or reduced. Activity against the single coyote cell population avail­ able was in most instances removed by these absorptions except when the absorbing cells were from dog 13. It was concluded that the anti­

genic mosaic of the cells from dog 13 was not as similar to that of

the individual coyote tested as that of the other cells used for

absorptions. Dog 13 cells did not, after 4 absorptions, remove all the

antibody specificity for the coyote's cells while doing so for a

second dog's (A-2) cells. When this antiserum was absorbed with coyote

12 liver cells, activity for one dog reactant in a panel (Table 11) was removed after 4 absorptions while cytotoxic reactivity for a

second dog's cells remained at a markedly reduced level. Thus a

sharing of alloantigens or the presence of cross-reacting antigens on

dog and coyote cells was definitely implied by the results of these

reciprocal absorptions. Viable cells from the coyote whose tissue was

used in the absorptions were not available for use in the cytotoxic

test.

A second dog anti-dog lymphocyte serum, represented in Table 12,

contained no reactivity for coyote 12 cells. In allogeneic fashion,

absorption of this antiserum with dog spleen removed absorbing cell

activity and removed or markedly reduced activity for a second dog's

cells in all but one instance. The absorbing cell in the latter case TABLE 9

COVOTE 12 ANTISERUM AGAINST DOG 3051 LYMPHOCYTES

REACTIVITY OF ANTISERUM AFTER 4 absorptions '3

ADSORBING CELLS REACTIVITY3 OF CELLS WITH UNABSORBED ANTISERUM D-l D-2 D-3 D-4 D-5 D-6 D-7 D-8 D-9 D-10 D-ll D-l 2

3051 . 556 16 8 8 8 32 16 32 64 64 32 32 64

3049 128 0 0 0 0 1 8 8 8 4 16 4 16

D-l 64 0

D-2 64 0

D-3 128 0

D-4 64 0

D-5 64 0

D-6 128 0

D-7 64 0

D-8 64 0

D-9 128 0

D-10 64 0

D-ll 64 0

D-l 2 64 0

Reciprocal of highest serum dilution showing greater than 2C% cell death in the lymphocyte cytotoxic assay. Absorptions performed with dog spleen cells. TABLE 10 DOG 3052 ANTIBODY AGAINST DOG 3051 LYMPHOCYTES

REACTIVITY OF ANTISERUM AFTER 4 ABSORPTIONS13

ADSORBING CELLS REACTIVITY3 OF CELLS VIITH UNABSORBED ANTISERUM D-l D-2 D-3 D-4 D-5 D-6 D-7 D-8 D-9 D-10 D-ll D-l 2 D-13 3051 512 32 32 16 32 64 128 64 64 128 64 128 128 !DC

3049 128 0 0 4 2 8 32 8 0 8 32 32 16 ND

Coyote 12 32 0 0 0 0 0 0 0 0 0 0 0 0 4

A-2 256 ND ID ND 1© ND to ID ID ID ID I'D ID 0

D-l 64 0

D-2 64 0 D-3 123 0

D-4 64 0

D-5 64 0

D-6 128 0

D-7 32 0

D-8 64 0

D-9 512 0

D-10 256 0

D-ll 128 0

D-l 2 16 0

D-13 1 ND

Reciprocal of highest serum dilution showing greater than 2Q£ cell death in the lymphocyte cytotoxic assay. ^Absorptions performed with dog spleen cells. cNot done.

I 90 91 was from dog number 10. Antibody activity against cells from the immunizing donor were not ever completely removed.

Dog Antibody Against Covote Cells: Renal Transplants

Clinical Observations

Ischemic time for the transplants ranged from 30 to 45 minutes.

(The kidneys were perfused with cold, heparinized lactated Ringer's solution.) The transplanted kidney in each case was filtering urine within 30 minutes of completing the vascular anastomosis. Rarely was there a dog that remained oliguric for the first 24 hours. In most instances a diuresis ensued for the next 24 to 48 hours. Forty-eight hours post-operative, urine excretion normalized until the onset of rejection. The leucocyte response in untreated and treated dogs was charted and the blood urea nitrogen (BUN) closely monitored. The renal grafted dogs were sacrificed in almost every instance a few hours prior to an agonal death. Each dog at that time was gravely depressed and had a BUN that was profoundly elevated. Many of the dogs were still excreting urine when sacrificed while others were anuric. A necropsy was performed on all experimental dogs.

Control Group

Six coyote kidneys were transplanted into untreated recipients.

Figure 7 depicts the leukocyte response of the 6 dogs in this group as an average of the composite data. The figure's purpose is to

illustrate the trend in leukocyte numbers before and after transplan­ tation. The partial hemogram depicted in the post-operative phase is consistent with a stressful experience and inflammation. The TABLE 11

DOG 3052 ANTISERUM AGAINST DOG 3051 LYMPHOCYTES

Animal Reactivity3 of Reactivity of Serum after Absorption Unabsorbed Serum with Covote 12 Liver 2x 4x

OP-4 512 512 0

OP-5 128 32 4

Coyote 12 32

3051 512

3 Reciprocal of highest serum dilution showing greater than 2Cp£ cell death in the lymphocyte cytotoxic assay.

92 TABLE 12

DOG 3041 ANTISERUM AGAINST DOG 3049 LYMPHOCYTES

REACTIVITY OF ANTISERUM AFTER 4 ABSORPTIONS13

ADSORBING CELLS REACTIVITY3 OF CELLS WITH D-l D-2 D-3 D-4 D-5 D-6 D-7 D-8 D-9 D-10 D-ll D-l 2 UNABSORPED ANTISERUM

3049 512 32 4 16 32 128 32 32 8 32 123 32 128

3051 64 0 2 1 0 16 1 4 2 4 64 4 8

Coyote 12 0

D-l 256 0

D-2 1024 0

D-3 0 0

D-4 16 0

D-5 16 0

D-6 32 0

D-7 1 0

D-8 64 0

D-9 0 0

D-10 0 0

D-ll 32 0

D-l 2 0 0

Reciprocal of highest serum dilution showing greater than 2C& cell death in the lymphocyte cytotoxic assay. ^Absorptions performed with dog spleen cells. immediate neutrophilic leukocytosis was an expected phenomenon follow­ ing such an extensive surgical procedure. The peripheral lymphocyte count became depressed in response to adrenal cortical secretion immediately following the surgery, and in general remained depressed below the normal base-line value until death. The average onset of detectable renal dysfunction as indicated by an elevated BUN greater than 40 mc$ was 4 days (range of 1 to 7 days). The BUN rose sharply in all dogs to an average terminal BUN of 282 m<$. The uremic syndrome is a common cause of a depressed lymphocyte count. The number of monocytes and eosinophilic granulocytes remained essentially normal.

The duration of renal function ranged from 8 to 21 days (mean of

10.8 days). The dog surviving the longest (21 days) in this group manifested signs of renal insufficiency the earliest, yet excreted urine until the day of death. Progressive tubular and glomerular malfunction were evident by the fourth post-operative day as indicated by the urinalysis findings of both a specific gravity within the range of the same molecular concentration as that of plasma dialysate and the passage of albumin. The long surviving dog (6182) was the only one of the 6 control dogs that had a naturally-occurring hemagglutinin titer (1:2) against donor erythrocytes. None of the control dogs had preformed cytotoxic antibodies against their donors. Five of the 6 transplants were performed across some degree of major erythrocyte antigen incompatibility (Table 21) without any detectable adverse effects on graft survival. xlCr Cells per cmm 0- 30 - - 6 2 28:- 2 2 4-14 - 18 - 20 24, ~ - 6 1 10 12 4 - 8 - i 0 6 2 - - - |- I Figure 7. Leukocyte response of the dogs in the Nothe Treatment in dogs ofthe response Leukocyte 7.Figure I I - ! - “

-4 6 \ ' \ ru. onspotdrpeeta vrg oftheaverage an represent plotted Points Group. composite data from this group.thisfrom data composite Jl ■2

0 • " / / I I Days I -- 2 4 Lymphocytes e. Neutrophils Seg. Leukocytes Total Transplantation 6

8 10 95 96

HADLG Treated Group

Each dog in this group received 5 subcutaneous injections of

HADLG (7 mg per kg of body weight) prior to transplantation. Figure

8 represents an average of the leukocyte response in these dogs.

Four of the 6 dogs (3039, 3042, 3045, 3040) experienced a mild inflammatory response to the heterologous protein injected by mani­ festing a neutrophilic leucokytosis. A lymphopenia was evident as early as 2 hours after the initial injection in all animals except dogs 3040 and 3042. In these cases, a moderate to profound lympho­ cytosis occurred subsequent to the HADLG administration. Both dogs were clinically healthy prior to treatment. Possibly this inconsis­ tent finding was a sequela to a preexisting condition that was unalterable by the usual effects of HADLG. Hemograms performed on dog 3042 three times a week for seven and one-half weeks prior to surgery revealed a peripheral lymphocyte count that undulated from three times greater than the normal value to a low normal value. In addition, immature neutrophils were observed in greater than normal numbers throughout this period. A profound regenerative left shift accompanying a leukocytosis was observed just prior to and during the first six HADLG injections. No obvious clinical condition could be ascertained to account for the "pathologic" hemogram in the apparent healthy dog. The lymphocyte count of both dogs eventually became markedly depressed. Post-operatively, the lymphocyte count generally remained considerably depressed in all dogs until death without the added effect of a uremic syndrome. As in the control group, a neutrophilic leukocytosis was evident the day following surgery but Start HADLG injections

Transplantation

Total Leukocytes

Neutrophils

Lymphocytes

2 ;~

Figure 8. Leukocyte response of the dogs in the HADLG treated group. Points plotted representof an average of the composite data from this group. 98 eventually the neutrophil count returned to near normal levels in the long term survivors. Eosinophilic granulocytes and monocytes remained in essentially normal numbers in the circulation.

Dogs 3040 and 3038 died of technical failures. Dog 3040 died subsequent to thrombosis of the renal artery. Dog 3038 was sacrificed on the third post-operative day because of pneumonia. A necropsy on this latter dog revealed a kidney that was grossly normal but surrounded by a hematoma.

Those dogs dying as a result of graft rejection had an average survival time of 38.7 days (range of 21 to 52 days). Three of the 4 dogs died in spite of daily HADLG therapy. Treatment was discontinued on dog 3045 at the forthieth post-operative day. At this time the dog was clinically healthy, with a normal BUN and excreting normal quan­ tities of urine. Six days after the last injection of HADLG the rejection process became evident as the BUN began to rise and the clinical manifestations of uremia ensued.

None of the dogs had preformed cytotoxins against their donor coyote. One of the dogs dying of a technical failure (3040) mani­ fested a hemagglutination titer of 1:8 against its coyote donor and two of the long term survivors (3039 and 3042) exhibited hemagglutina­ tion titers of 1:1 and 1:8 respectively against their donor's erythro­ cytes. All the transplants were performed across erythrocyte incom­ patibilities (Table 21); however, no acute transplant problems of an immunological nature were detected.

An indirect hemagglutination test was performed to determine if antibody specific for horse globulin had been produced by any of the dogs treated with HADLG. Table 13 illustrates the results of these tests. The test results depicted in Table 13 represent a reaction between tanned erythrocytes coated with horse globulin and serum collected at intervals after the initial HADLG injection. All of the control tests except one were negative at the starting dilution of

1:1000. The single positive control test was the reaction of normal

serum from dog 3042 with coated cells (1:256,000). This indication of

the presence of preformed antibody against horse globulin was further

substantiated by the apparent secondary response to the injection of

HADLG. All of the dogs treated with HADLG manifested a phenomenal

immune response to the heterologous protein. Rapid immune elimination

of the HADLG by combining with this anti-horse globulin was considered

to be the cause of loss in effectiveness in prolonging graft survival

in those animals rejecting their transplants concurrent with HADLG

therapy. Likewise, six days subsequent to discontinuing HADLG treat­

ment, dog 3045 showed evidence of considerable renal impairment.

For comparison to the HADLG-treated dogs receiving coyote kidneys,

a single dog-to-dog renal transplant was performed. This donor and

recipient were paired for allogeneic similarity as closely as possible.

Using the four antisera generated by skin graft immunization, a donor

(3049) was chosen for dog 9974 on the basis of cellular reactivity

depicted in Table 14. It is important to keep in mind, that these

typing sera are not monospecific and at most a reaction with the cells

of an individual indicates the sharing of antigens with the immunizing

donor. It does not in every instance reveal how many, if any, antigens

are not shared. It is very likely that reactivity against all of the TABLE 13

DOG ANTIBODY AGAINST HORSE GLOBULIN:

INDIRECT HEMAGGLUTINATION ASSAY

______TEST SAMPLE AND REACTIVITY13 Day3 Dog 3040 Dog 3012 Dog 3039 Dog 3045 Dog 3042

7 2000 2000 2000 8000 1024000

13 8000 32000 32000 64000 256000

22 64000 32000 64000 256000

28 512000 16000 64000

35 2048000 16000 64000

42 4096000 32000

49 32000

55 32000

58 4000 aNumber of days after the initial HADLG injection. t . Reciprocal of the highest serum dilution showing agglutination in the indirect hemagglutination assay. 100 101 canine histocompatibility antigens is not even represented in these four antisera. The antiserum WPUP anti-coyote 12 demonstrates activity for the cells of 3049 to a greater degree than it does with the cells of 9974. This could probably mean either a difference in antigen determinants or a difference in the amount of the same antigen present on the cell's surface. In addition, no erythrocyte antigen incompatibility existed nor did dog 9974 exhibit any preformed cyto­ toxic or hemagglutinating activity against the donor type. In addi­ tion to the daily 7 mg per kg dose of HADLG, three intravenous injec­ tions of normal horse globulin were administered. The latter was given at a rate of 75 mg per day for three days prior to the HADLG treatment. The use of the normal horse globulin was an attempt to induce a state of unresponsiveness in the treated dog to horse globu­ lin in an effort to prevent the rapid immune elimination of subsequent injections of HADLG.

The clinical data on this recipient (9974) is depicted in Figure

9. A lymphoblastosis was observed following the second normal globu­ lin injection. However, an acute lymphopenia was observed within hours after the initial HADLG injection. As seen previously, a neutro­ philic leukocytosis followed immediately after the HADLG injection.

Post-operatively, a profound neutrophilic leukocytosis ensued while the lymphocyte count diminished to zero. The BUN was normal at this time. Four days after survery, this dog manifested signs of acute gastritis and hemorrhagic enteritis. By the twelth day, after anti­ biotic and kaopectate therapy, the dog was progressing to a clinically healthy state. The lymphocytosis seen during the recovery phase u 16 “ v

14 “ ♦ NHG Injections V

12 “ Start HADLG injections

1 0 , ~ Transplantation 8 - i -Ji l i i — Total Leukocytes i 6 - i — Seg. Neutrophils 4 - Lymphocytes 2 -

i— r j i i___i__ i i 8 lcT 12~ 14 . 16 18 20 Days Figure 9. Leukocyte response of dog 9974 (A^CFHe) to HADLG treatment and transplantation with dog 3049 (A]CF) kidney. TABLE 14

HISTOCOMPATIBILITY TYPING3

ANTISERUM AND REACTIVITY13

WPUP Animal 3041 anti-3049 3052 anti-3051 Coyote 12 anti-3051 anti-Covote 12

3049 512 128 128 32

9974 512 128 128 4 aComparing cellular reactivity with a serum of unknown specificities.

Reciprocal of highest serum dilution showing greater than 2Cp6 cell death in the lymphocyte cytotoxic assay.

103 104

(Figure 9) was probably due to an increase in the circulating numbers of plasma-cell precursors. After the intestinal infection was con­ trolled, the numbers of these short-lived lymphocytes, diminished.

HADLG therapy was discontinued as a daily regimen at day 40 and con­ tinued every other day. The dog remained clinically healthy and an intravenous pyelogram performed on day 45 revealed a perfectly normal kidney. Four days later the BUN was at 60 m<$ and clinical manifes­ tations of uremia were evident. On the fifty-third day the kidney was greatly enlarged and readily palpable through the abdominal wall. The dog's life was prolonged for 12 additional days through the use of azathioprine and prednisone.

An indirect hemagglutination test on a single serum sample from this dog taken at day 22 revealed antibody against horse globulin at a level of 1:8,000 (Normal serum was not reactive with horse globulin).

This response was considerably lower than the responses observed on the same day in the other HADLG treated dogs. However, it was not known if the response was actually delayed due to the pretreatment with normal globulin or if the response was depressed for some reason associated with the acute gastro-intestinal illness the dog had pre­ viously experienced. At any rate, the dog began to reject the renal graft shortly after'the HADLG therapy was reduced as in the case with dog 3045.

Dog Anti-Poo and Covote Anti-Doci Lymphocyte Globulin Treated Group

Instead of using a heterologous ALG, homologous antilymphocyte

globulin was administered to 2 dogs receiving coyote kidneys and one 105 dog receiving a dog kidney. This was an attempt to prolong graft

survival without inducing antibody formation against the antilympho­ cyte globulin agent.

Dog OP-10 was matched incompatible with its donor (Coyote 12) by

serotyping as depicted in Table 15. Neither donor or recipient

reacted similarly with any of the typing sera. The antigenic differ­ ence between the animals as revealed by the first three sera (from

left) is of no consequence to the transplant since it appears that the

graft does not have antigenicity lacked by the recipient's tissue.

However, the reaction with serum WPUP anti-Coyote 12 indicates a strong

antigenicity not present in the recipient. An erythrocyte antigen

incompatibility existed between the donor and recipient. The dog had

a preformed hemagglutinin titer of 1:16 against the erythrocytes of

the coyote. In addition, the recipient dog manifested a preformed

cytotoxic antibody titer of 1:2 against the donor's lymphocytes and

the cells of two other dogs included in a panel.

The recipient was treated with anti-lymphocyte globulin (ALG)

(Coyote 12 anti-3051) at a dosage level of 7 to 10 mg per kg (alter­

nating) administered daily for two days prior to grafting and con­

tinued until death post-operatively. The antilymphocyte globulin of

course had no reactivity for Coyote 12 tissue, but reacted at a level

of 1:64 to 1:128 with the cells of dog OP-10 in the cytotoxic assay

and with a titer of 1:2 in the hemagglutination test.

The clinical data of this transplant is represented in Figure 10.

A lymphopenia was not observed at either two and one-half or 8 hours

after the second injection; however, a neutrophilic leukocytosis was TABLE 15

HISTOCOMPATIBILITY TYPING3

ANTISERUM AND REACTIVITY53

Coyote 12 WPUP Animal 3041 anti-3049 3052 anti-3051 anti-3051 anti-Covote 12 Coyote 12 0 32 0 1024

Dog OP-10 64 256 128 16 aComparing cellular reactivity with a serum of unknown specificities.

^Reciprocal of highest serum dilution showing greater than 2<$ cell death in the lymphocyte cytotoxic assay.

106 107 seen. The lymphocyte count was never depressed and even showed a trend toward being elevated during the last few days of life. The dog excreted urine in normal quantities post-operatively and was still excreting, although at a diminished rate, at the time of euthanasia on the eighth post-transplant day.

Dog A-l also received a kidney from Coyote 12. Table 16 illus­ trates the relationship between the two animals by serotyping. Again, as in the preceeding transplant, the major difference appears to be antigenicity expressed by the donor and not by the recipient. The recipient revealed no preformed cytotoxic or hemagglutinating anti­ bodies against the donor. There did exist an erythrocyte antigen incompatibility. Further, the coyote cells stimulated the recipient's cells to a degree of incorporating 2.1 times more tritiated thymidine than the recipient cells cultured alone. This was in comparison to a stimulation of 2.3 times when a dog's cells were incubated with a second dog's cells during the same culture period. Figure 11 depicts the hematological response to the treatment with antilymphocyte globulin (3041 anti-3049). This ALG did not react with the cells of the recipient in the hemagglutination test but was cytotoxic to a titer of 1:32 to 1:64 in the in vitro assay. No reactivity was directed against the cells of the transplant donor. Following the second intravenous administration of the ALG the lymphocyte count was depressed in this dog, although not below a normal value for the canine. Post-operatively, the lymphocyte count became elevated again.

The dog excreted urine until the day of death. The dog died within a matter of a couple of minutes following its last intravenous injection Start Coyote 12 anti-3051 ALG injections

Death

Transplantation

Total Leukocytes

Seg. Neutrophils

' Lymphocytes

-2 0 2 4 6 8 Days

Figure 10. Leukocyte response of Dog OP-10 (AjCFHe) to Homologous ALG treatment and transplantation with Coyote 12 (AgBCFTr) kidney. TABLE 16

HISTOCOMPATIBILITY TYPING3

ANTISERUM AND REACTIVITY13

Coyote 12 WPUP Animal 3041 anti-3049 3052 anti-3051 anti-3051 anti-Covote 12 Coyote 12 0 32 0 1024

Dog A-l 64 256 64 256

Comparing cellular reactivity with a serum of unknown specificities.

Reciprocal of highest serum dilution showing greater than 2Cp£ cell death in the lymphocyte cytotoxic assay.

109 xlOJ Cells per 2" " 32 0““ 30 “ 34 8““ 28 “ 6 3 12 “ 14 " 16 “ 18 0 2 22 - 24 “ 26 10 8 " - - ~ 1 I Figure 11. Leukocyte response of Dog A-l (CF) to Homologous ALG Homologous to (CF) A-l of Dog response Leukocyte 11. Figure I ”

\ / / / / Treatment and Transplantation with Coyote 12 (AgBCFTr) 12 Coyote with Transplantation and Treatment kidney. / / / / I Days * * Death Lymphocytes Neutrophils Seg. Start 3041 anti-3049 ALG anti-3049 3041 Start Total Leukocytes Total Transplantation injections

110

Ill

in an anaphylactoid-type manner. Prior to the injection, the dog was walking about. The dog had always reacted to the intravenous

injections with nausea and depression.

For comparison to the two dogs receiving coyote kidneys, a single

dog (3051) received a dog kidney (3052). Table 17 represents the

serotyping results. These four antisera in this instance revealed no

antigenicity in the donor that was not also present in the recipient.

The recipient's serum manifested no preformed cytotoxic or hemagglu-

tinating activity against the donor's cells. A slight incompatibility

in erythrocyte antigens did exist. In the mixed leucocyte culture

reaction, the donor's cells stimulated the recipient's lymphocytes to

incorporate 2.5 times as much tritiated thymidine as the control

culture.

The recipient was treated with 3052 anti-3051 ALG on the same

dosage schedule as the preceeding two dogs. The ALG did not hemagglu-

tinate the recipient's erythrocytes but was active in the cytotoxic

assay at a level of 1:256. Figure 12 illustrates the clinical data

gathered. A lymphocytosis was seen the day following the initial ALG

injection and the numbers of lymphocytes continued to increase for

several days. A lymphopenia was observed only on the day before death,

and could have been'due to an elevated BUN. Urine was excreted in

normal amounts for the first few post-operative days and then the

volume gradually diminished. The dog was still excreting small amounts

of urine at the time of death. TABLE 17

HISTOCOMPATIBILITY TYPING3

ANTISERUM AND REACTIVITY13

WPUP Animal 3041 anti-3049 3052 anti-3051 Coyote 12 anti-3051 anti-Covote 12 3052 32 0 32 8

3051 64 512 256 16 aComparing cellular reactivity with a serum of unknown specificities.

Reciprocal of highest serum dilution showing greater than 2C% cell death in the lymphocyte cytotoxic ass^y.

112 xlO Cells per 22 24_~ - 8 1 0 2 - 6 1 2 1 14 ~ 0 1 8 - 4 6 0 2_ , i — Figure 12. Leukocyte response of Dog 3051 (CF) to Homologous ALG Homologous to (CF) 3051 Dog of response Leukocyte 12. Figure “ - - - -

•2 / '

/ / 0 / / / Treatment and Transplantation with Dog 3052 (CFTr) 3052 Dog with Transplantation and Treatment kidney. / 2 / 4 6 “ “ — “ — Total Leukocytes Total — “ — “ “ 1 Days 10 8 * » Death Transplantation ALG Anti-3051 3052 Start Lymphocytes Neutrophils Seg. injections

113

114

HAPLG And Antigen Treated Group

Five dogs receiving coyote kidney transplants were treated with

HADLG and donor-specific spleen-cell antigen. The latter was an attempt to induce immunologic enhancement. All dogs were given 5 injections of HADLG prior to surgery and given daily injections post- operatively. Dog 3047 was pretreated with solubilized spleen cells injected intravenously three times a week for 13 injections ending on the day before transplantation (.807 mg per injection, 10.5 mg protein total). Two dogs (3046 and 3050) were pretreated with donor specific solubilized spleen cells injected intravenously three times a week for eleven injections ending on the day before surgery (.554 mg per injec­ tion, 6.10 mg protein total). Two dogs (3022 and 3024) were pre­ treated with 23 mg protein of donor specific solubilized spleen cells injected intravenously on the day before surgery. Post-operatively, these latter two dogs received 23 mg protein of solubilized liver cell antigen intravenously three times a week until death. Neither antigen preparation caused the dogs discomfort during administration.

Figure 13 illustrates the average hematological response of the dogs to the antigen pretreatment and the HADLG therapy. Uniformily a lymphopenia was observed after the initiation of the HADLG treatment.

Most of the dogs exhibited a neutrophilic leukocytosis. Except for an undulating peripheral lymphocyte count during the antigen administra­ tion period (not included in Figure 13), nothing unusual was seen in the hemogram during the pretreatment period. None of the dogs had preformed cytotoxic or hemagglutinating antibodies in their normal serum. Erythrocyte antigen incompatibilities existed between all / Start HADLG injections

Transplantation

_ _ Total Leukocytes

Seg. Neutrophils

Lymphocytes

12 14

Figure 13. Leukocyte response of the dogs in the HADLG and Antigen Treatment Group. Points plotted represent an average of the composite data from this group. 116 transplant pairs (Table 21). A cytotoxic and hemagglutination assay

on the serum of dogs 3046 and 3050 after ten antigen injections revealed no detectable antibodies against the donor coyote's cells.

This same information was not available for dog 3047.

Dog 3047 terminated as a technical failure. A necropsy revealed

occluding thrombi in both the renal vein and artery at the anastomatic

sites. Dog 3050 also died of a technical failure (thrombosed renal vein). Dog 3046 survived for 17 days. It received only one injection

of HADLG post-operatively (stock depleted). The excreted urine volume remained very good until the day of death; however, the rising BUN was evidence of massive tubular and glomerular damage. Dog 3022 and 3024

survived for 14 and 15 days respectively. Both dogs received HADLG

until the day of death. Urine excretion was good post-operatively but

gradually diminished after 10 days. The BUN in these latter 3 dogs

(3046, 3022, 3024) began to rise on or before the fifth post-operative day indicating early renal impairment. Serum collected from these

dogs just prior to death revealed antibodies against horse globulin.

The indirect hemagglutination titers manifested by the serum from the

individual dogs were as follows: 3047, 32,000; 3022, 64,000; 3024,

4,000; 3046 and 3050, 160,000.

Antigen Pretreatment Group

Dog 3044 was injected intravenously with donor specific solu­

bilized spleen cells three times a week for 13 injections (.807 mg per

injection, 10.5 mg protein total) prior to transplantation. A major

erythrocyte antigen incompatibility did not exist between this 117 transplant pair; however, the recipient had a naturally occuring hemagglutination titer of 1:8 against the donor's erythrocytes. No cytotoxic antibodies were detected. Post-operatively the kidney, excreted urine until the day before death. The dog with a BUN of

377 mc$ was sacrificed on the eighth day. Massive renal impairment was evident by the second day when the BUN began to rise. Figure 14 depicts the clinical data gathered on this dog. For a month prior to antigen treatment this clinically healthy dog manifested a rather stable lymphocyte count. During the period of antigen administration

(not included in Figure 14), the peripheral lymphocyte count undulated in a similar manner to the dogs in the previous antigen treated group.

Table 18 summarizes the average time of survival for the dogs in each group.

Serological Reactions of the Post Transplant Sera

Antiserum was collected from each dog recipient and analyzed by the lymphocyte cytotoxic and direct hemagglutination tests. Table 19 represents the reactivity of serum samples taken on or near the day of death with a panel of dog and coyote cells in the cytotoxic assay.

Only 7 different dog samples showed detectable antibody activity for either dog or coyote cells of the 19 different dog sera examined. It can be seen that 2 of the dog anti-coyote cell sera (5931 anti-coy­ ote 2, and 3047 anti-coyote 7) exhibit reactivity for dog cells in the absence of reactivity for coyote cells. Other sera show reactivity for some dogs and some coyotes but not for others. The first two (from left) serum samples were from untreated dogs, the third sample was xlC)3 Cells per o 30!“ 2 2 “ 26 14 “ 14 ~ 16 - 18 12 10 4 - 4 Q “ 8 6 2 Figure 14. Leukocyte response of Dog 3044 (A.£D) to Lymphoid Antigen Lymphoid to (A.£D) 3044 of Dog response Leukocyte 14. Figure - “ - “ - “ 642 0 -6-4-2 rtetetadTaslnainwt ooe6 (AjC) 6 Coyote with Transplantation and Pretreatment kidney. 2 4 6 Days 8 Death Lymphocytes e. Neutrophils Seg. Leukocytes Total Transplantation

118 TABLE 18

SUMMARIZED SURVIVAL DATA:

RENAL TRANSPLANTS

Mo Group Treatment HADLG3 Homologous ALG*3 HADLG & Antigen Antigen0

Donor Coyote Coyote Dog Coyote Dog Coyote Coyote Average Days of Survival 10.8 38.7 53 7 10 15.3 8 Range of Survival (Days) 8-21 21-52 6-8 14-17 aHorse anti-dog lymphocyte globulin.

Dog anti-dog or anti-coyote lymphocyte globulin.

°Donor specific solubilized spleen (and liver) cells.

119 TABLE 19

ANTISERA FROM DOGS WITH COYOTE RENAL GRAFTS: LYMPHOCYTE CYTOTOXIC ASSAY

ANTISERA AI€) REACTIVITY3 Dog 103 Dog 5931 Dog 3044 Dog 3047 Dog 3022 Dog 3050 Dog 3045 Anti Anti Anti Anti Anti Anti Anti Animal Covote 1 Covote 2 Covote 6 Covote 7 Covote 8 Covote 9 Covote 7

Dog 31M 2 32

Coyote 6 8

Dog 8 0

Coyote 3 0

Coyote 5 0

Dog 3052 0 0 >32 0 0 2 0

Dog A—1 0 0 4 0 16 2 4

Dog A-2 0 0 4 1 0 0 0

Dog 03 0 0 1 0 0 0 0

Dog 05 0 0 > 3 2 0 0 2 2

Coyote 12 0 0 8 0 8 2 1

Reciprocal of highest serum dilution showing greater than 2CpS cell death in the lymphocyte cytotoxic assay. from a dog receiving antigen treatment only, the fourth through sixth samples were from dogs in the antigen and HADLG treated group and the seventh sample was from a long term survivor receiving only HADLG.

The cytotoxic antibody demonstrated in the serum of dog 0P-10 prior to transplantation with coyote 12 kidney was not detected in the post­ transplant serum. Most likely the cytotoxic antibody had become adsorbed to the transplant.

Absorptions performed on one of these samples (3044 anti-coyote

6) with dog tissue and coyote tissue is shown on Table 20. Dog tissue was capable of removing all reactivity for the coyote's cells and reactivity for one other dog’s cells while leaving reactivity for certain other dog's cells. Absorption with coyote tissue was also capable of removing reactivity for one dog's cells and for the cells of the single coyote while leaving antibody activity for 2 other dog's cells.

Table 21 represents the hemagglutinating activity of the post- transplant sera. As can be seen 18 of 19 samples tested had antibody activity for dog and/or coyote erythrocytes. In several instancesj activity for dog cells was demonstratable in the absence of reactivity for coyote cells (105 anti-1, 5931 anti-2, 3012 anti-4, 3024 anti-8, and 3050 anti-9). There appears to be no correlation between the hemagglutinin response (Table 21) and the cytotoxic response (Table 19) thus indicating antibodies for different antigens.

In most instances, in which a preformed hemagglutinin titer existed, in brackets, the post-transplant titer was elevated. . The post-transplant hemagglutination titer of OP-10 became greatly 1 2 2

elevated. Allospecificity of the hemagglutinins in the dog sera was

not determined.

Immunofluorescence

A limited attempt was made to demonstrate erythrocyte antigens on

the surface of lymphocytes using monospecific dog anti-A^, C and D in

the indirect immunofluorescent test. These antisera were not cyto­

toxic to lymphocytes. Initially these antigens were demonstrated on

positive erythrocytes using the appropriate antiserum as shown in

Plate 8. Representative cells from each antigen type did show

fluorescence in the test combination. The peripheral beaded-pattern

of fluorescence was not very intense. Negative controls revealed no

fluorescence. Lymphocytes from dogs of the appropriate antigen type

exhibited fluorescence; however, negative controls also fluoresced.

No definitive conclusions could be drawn from these observations.

Canine Erythrocyte Typing

Coyote and dog erythrocytes were typed with monospecific antisera

prepared against dog erythrocyte antigens. Table 22 summarizes the

typing results using antisera against five major erythrocyte antigens

on 41 dogs and 12 coyotes. The most striking observations were:

(l) All coyotes tested were of the A 2 type as opposed to a 12.1 percent incidence of the same antigen in the dog population tested, and (2) Fifty-eight and one-half percent of the coyotes were of the

B type as opposed to an incidence of 4.86 percent observed in the dog population.

Table 23 shows the incidence of combinations of canine blood 123

Plate 8. Immunofluorescense of erythrocytes, from a C-antigen- positive dog, coated with mono-specific allogeneic anti-C (1:4 dilution) and rabbit anti-dog globulin conjugated with fluorescein isothrocyanate (1:5 dilution). TABLE 20

ANTISERUM FROM DOG 3044

GRAFTED WITH COYOTE 6 KIDNEY

Animal Reactivity3 of Serum Absorbed 3x With Unabsorbed Serum Doa A-l Cells Covote 12 Cells

Coyote 12 8 0 0

Dog 3052 > 32 > 3 2 8

Dog 05 > 3 2 > 3 2 > 3 2

Dog 03 1 0 0

Dog A—1 4

3 Reciprocal of highest serum dilution showing greater than 2($ cell death in the lymphocyte cytotoxic assay.

124 TABUE 21

ANTISERA FROM DOGS WITH COYOTE RENAL GRAFTS! DIRECT HEMAGGLUTINATION ASSAY

REACTANTS AND REACTIVITY3 Coyote Dog Dog Coyote Coyote Coyote Coyote Coyote Dog Dog Dog Dog Anti-Coyote 12 3038 31M 5 8 4 9 6 3052 A-l A-2 Antasera , (A2BC) (C) (BCD) (AjjBCD) (AjPC) (AjBC) (A^C) (A4C) (c ) (c ) (CD) 105 (CD) anti-1 ( A ^ r 0 0 0 0 0 4 8 103 (AXCD) anti-1 fA£) 0 0 0 2 0 1 4 5931 (A2BC) anti-2 (ApBCD) 0 0 0 2 4 2 6182 (AiC) anti-2 (A2B3D) 0 [2jc 6 4 [2] 12 8 0 0 2 3040 (CJ anti-3 (A2DC) 0 2 0 0 0 3039 (CD) anti-3 (A?BC) 4 [a 2 2 4 1 3012 (ECO) anti-4 (AjBC) 0 0 0 1 0 3043 (A,C) anti-5 (A2BCD) 0 0 4 1 1 1 2991 (C) anti-5 (ApECD) 8 rij4 4 8 8 3042 (AjC) anti-6 (A^O) 0 L4J4 0 1 0 3044 (A-£D) anti-6 (AnC) ?8 IS 4 2 ?*8 4 3047 (AjC) anti-7 (AjCD) 1 [ 1 8 2 8 8 3045 (CD) anti-7 (A43D) 1 4 0 1 1 3022 (AjC) anti-8 (A2DC) 8 2 8 1 3 0 2 4 (A^3) anti-8 (A-,BC) 0 0 0 . 1 3 0 4 6 (CD) anti-9 (Apfc) 1 1 2 1 3050 (C) anti-9 (A2BC) 0 1 1 OP-10 (AjC) anti-12 (A2BC) [if 512 4 8 1

Reciprocal of highest serum dilution showing agglutination. ) Encloses erythrocyte antigen type. ct 3 Encloses pretransplant hemagglutination titer. 1 2 6 group antigens in the two species. It is remarkable that the high incidence combinations seen in the coyote are the low incidence combinations in the dog.

Histopathologv

Light microscopy confirmed the diagnosis of immunological rejec­ tion of the renal transplants in the experimental dogs. Although the extent of parenchymal damage varied from dog to dog, some lesions were consistent in a given experimental group. Tissue samples for lesion evaluation were obtained at the time of euthanasia.

Plate 9 represents the appearance of a normal coyote kidney taken from a cortical biopsy. The most prominent lesion seen in the renal grafts of the untreated group was a mononuclear cell infiltration.

Plate 10 shows mononuclear cells marginating on the vascular endothe­ lium. The perivascular cuffing effect is dramatically illustrated here. In addition, tubular necrosis and albuminous casts in the tubular lumens are visible. Plate 11 again shows the intense mono­ nuclear cell reaction. The lumens of two arteries in the field are packed with nucleated cells and the interstitial spaces exhibit increased cellularity and edema (the latter being evidenced by the wide separation between the tubules). In addition, fibrinoid necrosis of the artery and glomerular solidification can be seen. The archi­ tecture of the renal parenchyma disentegrates from necrosis as ischemia from vascular disruption occurs.

The lesions exhibited by the rejected kidneys in the HADLG treated group were chronic-type changes. Most evident was Plate 9. Cortical biopsy of a coyote kidney showing a normal parenchyma. 128

Plate 10. Biopsy from a rejected coyote kidney in the no­ treatment group. Obvious lesions consist of perivascular cuffing of arteries (lower left and top center) and tubular casts (right). 129

Plate 11. Biopsy from a rejected coyote kidney in the no­ treatment group. Obvious lesions consist of edema, intense mononuclear-cell infiltration, fibrinoid deposition (homogeneous pink around artery - center left) and glomerular solidifi­ cation (center right). 130 interstitial fibrosis, nephron atrophy and apparent loss in numbers of nephrons, and proliferative vascular changes. Mononuclear cell infiltration varied in intensity but was in general less than in the no treatment group. Plate 12 represents the appearance of the general lesion in this treatment group. In addition to the prominent nephron atrophy with glomerular solidification and reduction in numbers of the same, an increased degree of interstitial cellularity, scattered deposits of fibrinoid material and a breakdown in the arterial adven­ titia with a build-up on the endothelial surface are evident. The lumen of the artery contains mononuclear cells, fibroblasts and occluding fibrin deposits. The tissue represented here is from a dog allograft (dog 9974 recipient of dog 3049 kidney). The lesions seen in the dog kidney did not appear to be different from those seen in dog rejected coyote kidneys. Plate 13 represents the coyote kidney from dog 3045. Atrophy of the glomerular tuft, interstitial fibrosis, mononuclear cell infiltration and arteriolar wall thickening are all present.

The grafts in the group receiving antigen and HADLG treatment were rejected in an accelerated fashion as indicated by their dura­ tion of function. Peritubular hemorrhage is most evident in every case along with mononuclear cell infiltration, interstitial edema, tubular necrosis with albuminous casts, and glomerular damage. Plate

14 shows these changes that are representative of lesions seen in all rejected grafts from this group.

The lesions manifested by the dog (3044) receiving antigen treatment alone were strickingly similar to those in the preceeding Plate 12. Biopsy from a rejected dog kidney in the HADLG- treated group. Obvious lesions consist of inter­ stitial fibrosis, nephron atrophy and degenera­ tive changes of the vasculature (artery - center bottom and glomerulus - top center). 132

Plate 13. Biopsy from a rejected coyote kidney in the HADLG- treated group. Obvious lesions consist of inter­ stitial fibrosis, slight amount of peritubular hemorrhage and atrophic glomerular changes. 133

Plate 14. Biopsy from a rejected coyote kidney in the HADLG and antigen-treated group. Obvious lesions con­ sist of massive interstitial hemorrhage and mono- nuclear-cell infiltration with tubular and glomerular necrosis. 134 group with vascular damage being very prominent as shown in Plate 15.

The artery depicted has undergone severe necrotizing damage and

ischemic necrosis of the tubules is evident.

The group treated with homologous ALG uniformily manifested

severe vascular damage. Interstitial hemorrhage and edema, tubular necrosis with casts and glomerular capillary damage were consistent

findings. Plate 16 illustrates the necrotizing vasculitis and glomer­

ular capillary thrombosis that was often seen. Mononuclear cell

infiltration was not as severe as in the untreated group. The pre­

sence of neutrophils in the vasculature and renal parenchyma was more

obvious than in any other group. 135

1

Plate 15. Biopsy from a rejected coyote kidney in the antigen treated group. Obvious lesions consist of fibrinoid deposition, massive hemorrhage and mononuclear-cell infiltration in the interstitinum. Tubular necrosis with casts and a necrotizing arteritis (lower left) are visable. Plate 16. Biopsy from a rejected coyote kidney in the homologous ALG treated group. Obvious lesions consist of severe necrotizing arteritis with occlusion (center bottom), glomerular capillary damage with fibrinoid deposition in Bowman's space (center top), tubular necrosis with casts (right), peritubular hemorrhage and mononuclear­ cell infiltration. 137

TABLE 22

DISTRIBUTION OF THE CANINE BLOOD GROUP ANTIGENS

Antigen Dogs3 , % Incidence Coyotes*3, % Incidence

41.4 AiJL 12.1 100.0 A2 B 4.86 58.4

C 97.57 100.0

D 21.92 25.0 aForty-one dogs jg Twelve coyotes 138

TABLE 23

II'C IDE ICE OF COMBINATIONS OF BLOOD GROUP ANTIGENS IN THE CANINE

Antigen Combination Dogs3 , % Incidence Coyotes*3, % Incidence > o 34.14 0

a 2 c 7.31 33.3

c 31.7 0

CD 9.75 0

Al CD 7.31 0 > o D 2.43 8.3 to

BCD 2.43 0 > 8 2.43 41.7 to

A 2 BCD 0 16.7

None detected 2.43 0 aForty-one dogs

Twelve coyotes DISCUSSION

This study was designed to evaluate the coyote and the dog as a model for xenogeneic transplantation. In the comparison of serum proteins from each animal type, no precipitation pattern unique to the dog or the coyote was found that would allow for a serological distinction between the two species. This was contrary to the findings in a previous report (Leone, 1956), in which the investigators based their conclusions on the differential amount of turbidity present in a tube precipitation test performed with the following reactants. The serum from a single dog and a single coyote were reacted with rabbit antisera prepared against each animal's serum. As demonstrated in the present study, rabbit antiserum against dog serum proteins will react against the sera from members of both species. It does so in a non- differentiating manner with varying degrees of strength as indicated by the variable numbers of precipitate lines and degrees of precipi­ tate density in the Ouchterloney test. The same was found to be true in the system employing rabbit anti-coyote serum. The differences in the reactivity of an individual's serum with either antisera was probably a reflection of the degree of similarity between the serum used for rabbit immunization and the immediate protein concentration in a given test sample. For instance, the same serum sample reacted with identical precipitation patterns in tests performed on different

139 140 days while two serum samples from the same animal collected on differ­ ent days did not always precipitate identically when reacted with the same antiserum on the same day. Absorption of both antisera with either dog or coyote serum always removed all activity for both dog and coyote serum.

It is true that using antiserum from a disparate species for the differentiation of two closely related species often fails to detect slight differences. It would be indicated to obtain antiserum from coyotes immunized against dog serum and vise versa in order to con­ clusively establish the antigenic relationship between the serum proteins of these two species. However, this was not performed since the cellular antigens were of more interest in this present study.

The heterologous lymphocyte sera represented by antibody pre­ pared in the horse and rabbit did not reveal any differences attri­ butable to species as shown by the complete removal of antibody activity against the cells of both species when the antisera were absorbed with lymphoid tissue of either the dog or coyote.

The difference in reactivity between the cells of the individual dogs and the coyote when reacted with the heterologous antisera pro­ bably reflected a modulation in the quantity or expression of cell

surface antigens due to cell cycle kinetics or serum factors which allowed the particular antigenic site(s) to be more or less available

for combination with antibody and complement.

The only difference between individuals of the two species that have been shown appear to be completely analogous to allogeneic type differences. The reactions of the homologous antisera with both dog 141:

and coyote cells and the absorption data presented on these antisera

demonstrated a sharing of cellular-antigenic determinents, most likely

alloantigenic determinents.

The antisera generated by skin graft immunization reacted with

the lymphocytes of several dogs and one coyote in an allogeneic

fashion. Proof of this allogeneic relationship was shown individually

by the dog anti-dog (3052 anti 3051), coyote anti-dog and dog-anti

coyote cell sera. Conclusive evidence showing that there was no

species-antigen difference also present on the cell surface of the dog

and coyote was not shown by the use of these antisera because of the

lack of available coyote cells for comparative reactivity. The dog

anti-dog (3052 anti 3051) cell serum could only manifest an alloantigen

response since all dogs possessed the species antigen. Conclusive

support for alloantigen sharing was demonstrated with absorbed 3052

anti-3051 antiserum. Reactivity for the coyote’s cells remained while

reactivity for the cells of dog A-l was abolished after the antiserum

was absorbed with cells from dog 13 (Table 10). Secondly, coyote

tissue absorbed from the dog antiserum, reactivity for one dog's cells while reactivity for a second dog's cells remained. The coyote anti­

dog and the dog anti-coyote also manifested alloantigenic reactivity.

This was shown by unabsorbed dog anti-coyote through its reactivity with the cells of both dogs and a coyote. In addition, absorption by

dog tissue removed activity for certain dogs while leaving activity

for others in the cytotoxic assay. Coyote anti-dog cell serum mani­

fested similar antibody activity against alloantigens. Absorption by

dog cells removed activity for certain dogs and not for others. 142

In addition to showing allogeneic activity, these latter two antisera had the potential for demonstrating the presence of species specific antigens if they existed as separate determinants on dog and coyote cells. In order to show this disparity, the cells of all coyotes would have had to react with the dog anti-coyote serum before and after absorption with dog cells. Similarly if the coyote anti­ dog serum would have shown reactivity for all dogs before and after absorptions with coyote tissue, a species specificity could have been concluded. Due to the availability of only one coyote for use in such an analysis, there was no conclusive evidence for or against a species specificity.

The coyote 12 anti-dog 3051 lymphocyte serum did in fact react with all dog cells tested. If more dogs had been tested, a non- reactor may have been found; or if another coyote had been immunized an antiserum with a more limited range of specificity may have been generated. It is interesting to note that this coyote (12) apparently had a minimal number of allotypes but with a very common representa­ tion. Table 6 shows that coyote 12 anti-dog 3051 reacted with 36/36 dogs included in a panel. In the absorptions of 3052 anti-3051 illustrated in Table 10, activity for coyote 12 cells was removed by the cells of 12/13 dogs used. Dog 3041 anti-dog 3049 cell serum was not cytotoxic for coyote 12 cells at all. Absorption of 3041 anti-

3049 with coyote tissue was indicated (but not performed) to show definitively that the antiserum had no activity for the coyote.

Interestingly enough, 3041 anti-3049 was not cytotoxic in the in vitro assay for the cells of dog 10, yet this dog's cells absorbed activity 143 from the antiserum as measured by a reduced reactivity with dog 3049 cells. This is one of several examples in this study in which nega­ tively reacting cells were able to adsorb antibody activity. This is most probably an example of the phenomenon frequently observed in human histocompatibility testing and referred to as CYNAP (cytotoxic negative, absorption positive) (Ceppelini, 1965).

The existence of alloantigens shared by humans and chimpanzees has been shown (Zmijewski, 1965) (DeWitt, 1970). Antisera useful for this purpose are human anti-human, chimpanzee anti-chimpanzee and chimpanzee anti-human cell serum. However, the human and chimpanzee also have a cellular antigen(s) unique to each. This species-specific antigen can be demonstrated in the chimpanzee anti-human cell serum after absorptions by chimpanzee cells. Human anti-chimpanzee cell serum manifests only the species-specificity.

Conclusive evidence that the dog and coyote do not differ in species-specificity from one another to the degree that man and the chimpanzee do was clearly shown by the reactivity of the post-trans- plant sera before and after absorptions. The unabsorbed dog anti­ coyote cell sera (Tables 19 and 21) showed excellent reactivity in some instances for certain dog's cells in the absence of detectable activity for coyote cells. Furthermore, absorption of the dog 3044 anti-coyote 6 cell serum (Table 20) with dog A-l cells removed reactivity for the cells of a coyote and a certain dog while leaving activity for two other dog's cells in the cytotoxic assay. Thus, only allogeneic-type differences could be demonstrated. The range of survival times (8 to 21 days) in the untreated group was indicative 144 itself of an allogeneic transplantation model. Shanfield and asso­ ciates (1968) reported similar survival times in dog renal allograft­ ing.

The present findings and the observations made by Bull (1971) that the coyote shares all the known dog erythrocyte antigens except for the A-^ antigen, lends support to a close relationship between this pair. The peculiarity of the coyote in possessing a perfect incidence of the antigen is not easily explained, but may be contributed to by the low incidence of existence, if at all, of coyote varieties (there are many varieties of dogs) (Young and Jackson, 1951). Taxonomically, the coyote is considered to be a direct ancestor to the dog and the

antigen is thought to be the parent to the more common A-^ antigen found in the dog population (Bull, 1971).

Further support for a true allogeneic relationship between this pair comes from karyotyping studies. Both animals have a diploid number of 78 and the karyotype of the coyote is indistinguishable from various races of the domestic dog (Hsu and Benirschke, 1967). The dog and coyote do interbreed and the progeny are fertile (Young and

Jackson, 1951).

The role of the erythrocyte antigens in canine transplantation has only been sparsely studied. Altman (1965) evaluated the A^ group in canine skin and renal allotransplantation. Transplanted tissues were from animals of the A^ antigen type. Altman transplanted across this antigen incompatibility; he transplanted into recipients actively immunized against the A^ antigen; and he perfused kidneys and skin graft beds with potent anti-A^ serum. No decrease in graft 145. survival over control graft survival was observed in these studies.

In agreement with the findings of Altman, the work of Rapaport and associates (1971) has shown no direct evidence that incompati­ bilities for antigens A, C and D have an adverse influence upon the immediate duration of cardiac allograft survival. The recipients were unmodified canine hosts closely matched with their donors for the leukocyte antigens (DL-A). The average survival was 37.5 days.

Rubinstein and Ferrebee (1964) reported that reactivity for erythrocyte blood group antigens A and D could not be absorbed from monospecific antisera by tissues or by leukocytes from animals carry­ ing the A or D antigens on their erythrocytes. The sera were only absorbed once for 12 to 24 hours at 3 7 ° C with 10® or 10^ leukocytes per 0.8 ml of serum. The leukocytes used for the absorptions were grossly contaminated v/ith erythrocytes. The average ratio of leuko­ cytes to erythrocytes was 2:1. This number of contaminating erythro­ cytes could have surely reduced the hemagglutinin titer of the speci­ fic antiserum if more absorptions had been performed. A purer leukocyte preparation and additional absorptions were indicated for a definitive conclusion on the presence or absence of the A and D antigens on leukocytes. Coomb's mixed cell agglutination technique was tested with epidermal cells and with leukocytes for both anti-A and anti-D sera. The reported results were negative. The attempt to demonstrate erythrocyte antigens A^, C and D on the surface of lympho­ cytes by fluorescence in this present study gave no conclusive evi­ dence one way or the other. The test lymphocytes did fluoresce as did the control cells. This non-specific fluorescence may have been 146 abolished by using a greater dilution of conjugate or by attempting a direct fluorescent antibody test.

Puza and associates (1964), Rubinstein and Ferrebee (1964) and

Rubinstein and associates (1968) studied the allohemagglutinins

(erythrocyte agglutinating antibodies) elicited by skin grafting and homologous tissue inoculation in the dog. Lymphocyte cytotoxic anti­ bodies were simultaneously generated but their reactivity did not correlate with the reactivity of the hemagglutinins. These studies suggested a similarity in certain antigens of skin, erythrocyte and leukocyte in this species. Eleven allogenic-immune sera were tested against the erythrocytes of 40 dogs. The reactions observed did not conform to those expected on the basis of the canine blood group antigens A, C and D.

In further studies (Rubinstein ert al.., 1963) it was shov/n that the reactivity of the 11 allo-hemagglutinins could be absorbed by tissue and by leukocytes. Some evidence was presented to show that there might be a slight decrease of skin allograft survival when donors had certain of the allo-hemagglutinogens lacked by the reci­ pient. Further identification of the antigens responsible for these erythrocyte agglutination reactions or their relationship to the DL-A antigens has not been attempted by anyone.

The coyote-to-dog transplants reported here elicited hemagglu­ tination responses in 18 of 19 animals evaluated. Identification of the specificities of these hemagglutinins was not attempted. The animals under study were typed only for the A^, A2, B, C and D allo- antigens. The blood types of the graft donor and recipient as well as 147

the type of the reactant's cells are included in Table 21. An examina­

tion of these results does not allow even for a generalization on the

specificity of the reactions recorded. For example, dog 5931 (type

A 2BC) received a kidney from coyote 2 (type A 2BCD). An antibody

specificity against the D antigen would be expected. Rubinstein and

associates (1968) in their skin graft studies showed that only one

classic blood group antibody, anti-O, was elicited, by grafting, in

titers that allowed positive identification. Only the A, C, and D

antigens were investigated. As can be seen from Table 21, the resul­

tant serum did not react with two D-positive reactants but did react with one D-positive and 2 D-negative dogs. This type of inconsistency

shown by several other sera indicated reactivity against a minor blood

group antigen not included in the cell typing panel or more likely a

reaction against a cell antigen shared by both erythrocytes and kidney

tissue. The latter is reminescent of the reports by Rubinstein and

associates (1964, 1968) on hemagglutinins generated by skin grafting

in the dog.

The importance of the DL-A antigens in canine transplantation is without question (Dausset al.., 1971). Closely matched siblings and

non-siblings survived longer after renal and cardiac allografting than

did mis-matched recipients. The same was true in other species

studied. There is adequate evidence from the literature that antigens

capable of stimulating transplantation immunity can, under special circumstances, prolong allograft survival. In the rat (Guttmann et al..

1972), the presence of specific antibody to transplantation antigens often prolongs renal graft survival. The antibody can be generated 1<48 by active immunization or administered passively. Similar reports of enhancement have been made on mouse skin grafts (Chutna, 1971). Smith

(1971) was successful in prolonging dog renal allograft survival by the use of passively administered allogeneic antibody (pepsin digested

IgG). The dog anti-dog kidney globulin was produced by renal grafting with additional inoculations of kidney stroma with Freund's adjuvant.

The anti-kidney globulin was slightly toxic to lymphocytes and showed a leukoagglutination titer of 1:256. Horse anti-dog lymphocyte globu­ lin was an adjunct treatment. Untreated animals survived for 11.5 days. Animals treated with HADLG plus the anti-kidney globulin sur­ vived for an average of 24.8 days. The DL-A type of the recipient and the specificities of the anti-kidney globulin were not considered.

The prolonged graft survival was attributed to immune enhancement by the anti-kidney globulin rather than to an additional immuno­ suppressive effect.

If enhancement was the mechanism of graft survival prolongation in Smith's study (1971), the protective antibody could have been coating the vascular endothelium. Vetto and Burger (1971) have shown that vascular endothelium derived from either kidney, liver, or a musculoskeletal bed contained antigenic determinants demonstrated by a lymphocyte alloantisera test panel in both the dye exclusion cyto­ toxicity method and the chromium-51 isotope release method. Further­ more, the antigens contained on these various endothelial cell

sources in the same animal often vary among themselves. There was almost always a difference in antigenic profile between an endothelial cell and its corresponding lymphocyte. These differences indicate 149 that the expression or detection of a valid histocompatibility profile may not be possible by the serotyping of a single cell. The concept of determining antigens beyond those found on lymphocytes is being used clinically by DePlanque and associates (DePlanque, 1969). These investigators demonstrated allogeneic typing antiserum reactivity with kidney cells in the immune adherence assay in the absence of reac­ tivity against lymphocytes and visa versa.

Not only is the knowledge of the antigenic profile of a trans­ planted organ necessary for proper matching of donor and recipient, it is also essential to an enhancement or tolerogenic scheme. Immuni­ zation of a host with specific transplantation antigens for the pro­ duction of enhancing antibodies has advantages over the use of passively administered antibody. In the former, active participation of the graft recipient exists. There is a continuing production of serum antibody provided in part by the stimulus of antigens in the graft. Passive enhancement, on the other hand, seems to involve the specific inhibition of all responsiveness by serum antibody.

The effects of antigen pretreatment can be modified by altera­ tions in certain variables such as source and form of antigen, antigen dose, and route and timing of antigen administration. Wilson (1969a) observed a prolongation of graft survival after the use of subcellular splenic antigen in combination with azathroprine and methyl predniso­ lone (average survival 144 days). No prolongation of graft survival was seen with the use of the two immunosuppressive agents used alone, and antigen used alone caused accelerated rejection. This prolonged graft survival was explained as being a result of-the..enhancement 150 phenomenon, or the generation of non-cytotoxic antibodies that coated the graft and prevented its destruction by host-immune mechanisms.

When HADLG was combined with spleen antigen, administered as before,

Wilson observed a range of survival from 15 to 28 days. Using a dose of HADLG (7 mg/kg body wt.) that was twice the dose used by Wilson, graft survival was prolonged to a range of 15 to 17 days in this present study. The grafts were rejected, however, in an accelerated fashion, in that the survival time was much less than when HADLG was used alone and histological evaluation revealed in large measure the participation of humoral antibody in the rejection as evidenced by the severe vascular damage manifested. The lesions were comparable to those seen in the rejected kidney from the dog receiving antigen only.

After the antigen pretreatment, circulating cytotoxic antibodies were not detected in the serum of the recipients in this study or in

Wilson's report. Earlier studies (Kronman et al.., 1966) indicated that canine skin graft survival was prolonged after recipient pretreatment with donor specific cellular antigen if the graft was applied before the appearance of cytotoxic antibody.

In explanation of the observed patterns of immune response seen in Wilson's study and the work reported here, the low dose antigen pretreatment possibly stimulated the production of a non-cytotoxic antibody and at the same time primed the host for the production of complement fixing antibodies that would appear in a secondary response following transplantation. Treatment with the chemical immunosuppres­ sive agent was capable of reducing or abrogating this humoral cytotoxic immune response while treatment with HADLG failed to do so. It is documented in the literature that heterologous ALG is more effective

in diminishing the cell-mediated responsiveness than the humoral anti­

body response; showing even less effect in modifying the secondary

humoral immune response (Sell, 1969). The addition of liver antigen

to the treatment regimen was without noticable beneficial effect.

Caine (1971) has reported the survival of renal allografts in adult

swine for up to eleven months after being treated at the time of

transplantation with donor specific solubilized liver antigen. When

this treatment protocol was used in dog and monkey grafting experi­

ments, results were unimpressive. Caine presented the proposition

that pigs possibly vary in the amount and/or type of transplantation

antigens produced by the liver so that a tolerogenic or immune enhance­

ment effect was more easily induced in the swine model.

There are few reports of the use of allogeneic antiserum against

cellular antigens in transplantation studies for the purpose of immuno­

suppression. Ono and associates (1969) reported prolonging the sur­

vival of rat cardiac allografts after treatment with antiserum

against the recipient's antigen type. Taub (1969) has demonstrated

the prolongation of skin allograft survival by recipient-specific

mouse-antimouse thymocyte antiserum. Both papers reported a lympho­

penia. Taub suggests that the most probable mechanism of immuno­

suppression by alloantisera would be lymphocytotoxicity resulting in

depletion of lymphocytes. No prolongation of renal graft survival was

seen in this present study and only a transient lymphopenia could be

shown. The histological lesions observed indicated an accelerated

rejection. The antisera were recipient specific. No cytotoxicity 152 was demonstrated with the lymphocytes of the graft donor. The vascular damage revealed on histology, the presence of neutrophils and only a moderate mononuclear cell infiltration, and the short duration of graft survival implied a prominent role of humoral antibody in destruction of these grafts.

The renal lesions due to humoral antibody could have been caused,

first of all, by complement-fixing antigen-antibody complexes depo­

sited non-specifically in renal capillaries. The administered allo- antibody in this case combining with soluble alloantigens or platelets

in the serum of the host together with or without complement compo­

nents. The release of vaso-active substances as a result of this

combination of factors v/ould promote an increase in vascular permea­ bility with resultant immune complex deposition on the limiting membrane of the vessels through a process of filtration. This depo­

sition together with the accumulation of leukocytes and their libera­

tion of enzymes would lead to destruction of the vessel wall

(Cochrane, 1971) and (Henson, 1971). A similar pathogenesis can be

seen in Systemic Lupus Erythematosis (SLE). Renal involvement in SLE

is primarily glomerular but may at times be associated with a necro­

tizing vasculitis of interlobular arteries and afferent arterioles

resulting in acute ischemic changes of the renal tubules (Golden and

Maher, 1971).

A second mode of renal damage mediated by humoral antibody could

have been an interaction of immunoglobulin and specific graft antigen.

The passively administered antiserum was not cytotoxic to the reci­

pient's lymphocytes in the in vitro assay; however, the antigen may 153 have been on the lymphocyte in such an antigenic mosaic that it was cytotoxic negative but absorption positive. The indicated absorp­ tions were not performed to verify this.

Alternatively, the antigen may have been absent from the lympho­ cyte's surface but present on other nucleated cells of the graft donor as discussed earlier (Vetto and Burger, 1971). The passively adminis­ tered antibody may have been directed at an antigen shared by kidney and immunizing skin cells. In concert with this latter hypothesis,

Cerilli and associates (1971) have reported a non-complement dependent antibody directed against vascular endothelium and demonstrated by the indirect fluorescent antibody technique that was responsible for graft damage. This antibody in the pretransplant serum of patients was not detected by conventional typing and matching procedures. The antigen against which the antibody was directed appeared to be absent from the lymphocyte's surface, for repeated lymphocyte absorptions of a serum showing both cytotoxicity and positive immunofluorescent antibody activity removed only the cytotoxic activity.

The allogeneic antilymphocyte globulin used in this study was not immunosuppressive in the dog. The antibody was directed at allo- antigens and it is suggested that the lack of preferential in vivo reactivity with lymphocytes resulted in its failure to be immuno­ suppressive. In accordance with this suggestion, the cellular and organ distribution of alloantigens in the dog may not be similar to that seen in the mouse or rat in which allogeneic antisera are immunosuppressive. It is suggested that alloantigens throughout the dog's body adsorbed the passively administered antibody before it 154 could influence the lymphocyte population. In addition, if there was no specificity for the donor tissue, an enhancement phenomenon, as proposed by some (Guttmann et .al., 1967) to play a role in heterolo­ gous ALG mediated graft survival, along with the immunosuppressive effect, could not exist.

Alternatively, the allogeneic antilymphocyte globulin may have been inherently incapable of immunosuppression. By using a different immunization regimen, antibodies capable of prolonging graft survival may have been generated. SUMMARY

The dog and coyote were studied as a possible model for xeno­ geneic transplantation. The following sera were used to evaluate the antigenic differences between these two canine types.

1. Rabbit antisera against dog or coyote sera

2. Horse antiserum againstdog lymphocytes

3. Rabbit antisera against dog or coyote lymphocytes

4. Dog antisera against dog or coyote lymphocytes

5. Coyote antiserum against dog lymphocytes

The Ouchlerlony, Immunoelectrophoresis, lymphocyte cytotoxicity and direct erythrocyte hemagglution techniques were employed for the evaluation of the antisera with appropriate antigens. Serological differences revealed by the reactions of the heterologous and homo­ logous antisera appeared to be only allogeneic-like differences without a species disparity being found.

The function of the renal transplants and the host's immunologic response to the graft was not noticably different in the coyote-to- dog model from the dog-to-dog model. The survival times observed

(8-21 days) for coyote-to-dog renal grafts were like the survival

l times reported for dog renal allografts.

Graft survival was prolonged by the use of heterologous (horse)

ALG; however, an attempt to prolong graft survival by host

155 156 immunosuppression with homologous ALG (dog anti-dog and coyote anti­ dog) was unsuccessful. The induction of immunologic enhancement of graft survival was not achieved. Host sensitization to transplant antigens resulted from recipient pretreatment with donor specific solubilized lymphoid cell antigen in the attempt to induce immuno­ logic enhancement.

Information obtained from this study indicated that the coyote and dog did not represent a suitable model for xenogeneic trans­ plantation. BIBLIOGRAPHY

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