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The effect of the W gene on immune responses in mice McNay, Margaret Lynne 1975

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THE EFFECT OF THE W GENE ON IMMUNE RESPONSES IN MICE by MARGARET LYNNE McNAY B.Ed., University of British Columbia, 1969 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of Zoology We accept this thesis as conforming to the require^ standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1975 In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th i s thes i s for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t i on of th is thes is for f i nanc ia l gain sha l l not be allowed without my written permiss ion. Department of ~~**?f n^ J:rcf^~ The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date L SkJ-*l*^ ABSTRACT Previous reports that lymphocytes from W/Wv mice migrate more slowly than do lymphocytes from normal mice, and that spleens from W/WV mice produce fewer plaque-forming c e l l s i n response to immuni-zation with sheep red blood c e l l s (SRBC) than do normal mice, suggested that other immune responses i n these mice might also be a f f e c t e d . The following experiments have revealed that g r a f t r e j e c t i o n i s f a s t e r and more vigourous among W/WV mice than among +/+, normal mice: 1) strongly antigenic, H-2 incompatible, s k i n a l l o g r a f t s examined h i s t o -l o g i c a l l y at the eighth day a f t e r transplantation to W/WV hosts e x h i b i t higher g r a f t r e j e c t i o n scores than do i d e n t i c a l s k i n a l l o g r a f t s trans-planted to +/+ hosts; 2) weakly antigenic skin a l l o g r a f t s bearing the H-Y antigen, when trans-planted to female +/+ and W/Wv hosts, are rejected more quickly by W/WV hosts than by +/+ hosts; 3) the uptake of t r i t i a t e d thymidine by c e l l s i n the p e r i p h e r a l blood of mice bearing skin a l l o g r a f t s indicates a greater proportion of a c t i -vated c e l l s i n the c i r c u l a t i o n of W/WV mice than of +/+ mice. Confirmation has also been obtained that some antibody responses among W/WV mice are depressed. In response to immunization with SRBC, W/WV mice produce about 1/4 the number of plaque-forming c e l l s as do +/+ mice. W/+ and WV/+ mice produce intermediate numbers of plaque-i i forming c e l l s . T i t e r s of SRBC agglutinins produced by W/W mice i n a secondary response are s i g n i f i c a n t l y lower than t i t e r s produced by +/+ mice. The uptake of t r i t i a t e d thymidine by spleen c e l l s stimulated v/ith the B - c e l l mitogen LPS indicates that W/WV c e l l s are deficient: i n t h e i r response to t h i s mitogen. Treatment with the T - c e l l mitogen Concanavalin-A, however, r e s u l t s i n marginally greater thymidine uptake by W/WV spleen c e l l s than by +/+ c e l l s . These r e s u l t s suggest that T - c e l l responses generally may be somewhat enhanced and B - c e l l responses v depressed i n mice of W/W genotype. The a l t e r e d r e a c t i v i t y of lymphocytes from W/WV mice can be accounted f o r by p o s t u l a t i n g an a l t e r a t i o n i n the surface c h a r a c t e r i s -t i c s of these c e l l s . The hypothesis that the W locus controls a c e l l surface c h a r a c t e r i s t i c i s the only hypothesis yet formulated which i s able to account f o r a l l the better-known p l e i o t r o p i c e f f e c t s of the W mutation (anemia, s t e r i l i t y , and a lack of pigmentation) as w e l l as for i t s immunological e f f e c t s . This hypothesis and i t s implications are discussed i n d e t a i l i n the text. TABLE OF CONTENTS Page ABSTRACT . • • • 1 1 LIST OF TABLES i x LIST OF FIGURES x i LIST OF ABBREVIATIONS x i i ACKNOWLEDGEMENT . x i v INTRODUCTION • • • 1 MATERIALS AND METHODS 11 I. MICE 11 II. SKIN GRAFT STUDIES . 11 A. Transplantation Technique 12 B. Measurement of A l l o g r a f t S u r v i v a l 13 C. Establishment of C r i t e r i a by Which to Measure A l l o g r a f t S u r v i v a l 16 1) Iso g r a f t Studies 16 2) A l l o g r a f t Studies 17 a) Determination of h i s t o l o g i c a l c r i t e r i a f o r assessing a l l o g r a f t s u r v i v a l 17 b) Determination of MST of DBA/2 gr a f t s on C57B1/6 hosts 18 D. A l l o g r a f t Rejection i n W/WV Mice: Grafts Exchanged Across H-2 B a r r i e r s 18 E. A l l o g r a f t Rejection i n W/WV Mice: Grafts Exchanged Across Weak H i s t o c o m p a t i b i l i t y B a r r i e r s 20 F. H i s t o l o g i c a l Preparation of Skin Grafts 21 i v V Page I I I . LYMPHOCYTE ACTIVATION STUDIES . . . . 21 A. L a b e l l i n g iri vivo 22 B. L a b e l l i n g i n v i t r o 22 C. S c i n t i l l a t i o n Counting . . . 23 IV. HUMORAL IMMUNE RESPONSES 24 A. Production of SRBC Agglutinins i n +/+ and W/WV Mice . 25 B. Production of Plaque-forming C e l l s i n +/+, W/+, WV/+, and W/Wv Mice 26 V. MITOGEN STIMULATION STUDIES 28 VI. OTHER STUDIES . . . . 31 A. Macrophage Migration Studies 31 B. Lymphocyte Adhesion Studies 32 VII. HAEMATOLOGICAL TECHNIQUES 36 RESULTS 37 I. SKIN GRAFT STUDIES 37 A. H i s t o l o g i c a l Features of Skin Isograft Healing . . . 37 B. H i s t o l o g i c a l Features of Skin A l l o g r a f t Rejection . . 41 1) General Observations 41 a) The h e a l i n g - i n phase 41 b) The acute r e j e c t i o n phase 42 c) The replacement phase 52 2) H i s t o l o g i c a l C r i t e r i a for Assessing Degree of A l l o g r a f t S u r v i v a l 52 3) MST of DBA/2 Skin A l l o g r a f t s on C57B1/6 +/+ Mice 60 C. A l l o g r a f t Rejection i n W/WV Mice 60 1) On the Eighth Day A f t e r Transplantation . . . . 60 v i Page 2) MST of DBA Skin A l l o g r a f t s on +/+, W/+, WV/+, W/WV Hosts 60 3) Grafts Exchanged Across Weak H i s t o c o m p a t i b i l i t y B a r r i e r s .64 I I . LYMPHOCYTE ACTIVATION STUDIES 66 A. L a b e l l i n g i n vivo 66 B. L a b e l l i n g i n v i t r o 66 I I I . HUMORAL IMMUNE RESPONSES 70 A. Production of SRBC Agglutinins i n +/+ and W/WV Mice . 70 B. Production of Plaque-forming C e l l s i n +/+, W/+, WV/+, and W/Wv Mice 73 IV. MITOGEN STIMULATION STUDIES 75 V. OTHER STUDIES 78 VI. A. Macrophage Migration Studies 78 B. Lymphocyte Adhesion Studies 79 VI. HAEMATOLOGY . 80 A. Ungrafted Mice 80 B. Mice Bearing Skin Autografts or Skin A l l o g r a f t s . . . 81 DISCUSSION 83 I. TRANSPLANTATION IMMUNITY 83 A. Genetics of Transplantation 83 1) C h a r a c t e r i s t i c s of Transplanted Tissue 83 2) H i s t o c o m p a t i b i l i t y Genes 86 3) Other Genes A f f e c t i n g Immune Responses 87 B. Recognition and S e n s i t i z a t i o n 88 1) Antigen Recognition by Lymphocytes 88 2) S e n s i t i z a t i o n 89 v i i Page C. The Rejection Process 94 1) Role of Lymphocytes 94 2) The C e l l u l a r I n f i l t r a t e 95 3) E f f e c t o r Mechanisms i n A l l o g r a f t Rejection . . . 98 a) S p e c i f i c i t y of g r a f t r e j e c t i o n 98 b) Contact-induced cell-mediated c y t o t o x i c i t y . 99 c) Role of lymphokines 100 d) Role of antibody 102 e) Conclusion 103 D. Skin A l l o g r a f t Rejection i n W/WV Mice 104 1) MST of F i r s t - s e t H-2 Incompatible Grafts . . . . 104 2) Graft Rejection at the Eighth Day A f t e r Trans-p l a n t a t i o n 104 3) Rejection of Skin A l l o g r a f t s Across Weak H-B a r r i e r s 106 4) A Study of the C e l l u l a r I n f i l t r a t e (Collen, 19 74) 106 I I . LYMPHOCYTE ACTIVATION STUDIES 107 A. General Discussion 107 B. Lymphocyte A c t i v a t i o n Studies i n +/+ and W/WV Mice. . 110 C. A C o r r e l a t i o n of Observations from Lymphocyte A c t i -vation Studies and A l l o g r a f t Rejection Studies i n +/+ and W/Wv Mice 110 I I I . HUMORAL IMMUNE RESPONSES 114 A. General Discussion 114 v B. Humoral Immune Responses i n +/+ and W/W Mice . . . . 118 IV. MITOGEN STIMULATION STUDIES . . . 120 A. General Discussion 120 B. Mitogen Stimulation Studies i n +/+ and W/WV Mice . . 125 v i i i Page V. OTHER STUDIES 127 A. Macrophage Migration 127 B. Lymphocyte Adhesion . . • 127 VI. HAEMATOLOGY 129 VII. SPECULATION: THE PRIMARY SITE OF ACTION OF THE W GENE . . 130 CONCLUSION 138 REFERENCES 139 LIST OF TABLES Table Page I. H i s t o l o g i c a l features of s k i n i s o g r a f t healing . . . . 38 I I . H i s t o l o g i c a l features of s i x stages of skin a l l o g r a f t r e j e c t i o n i n normal mice 58 I I I . Graft r e j e c t i o n scores determined by h i s t o l o g i c a l analysis of DBA/2 skin a l l o g r a f t s on +/+ hosts . . . . 61 IV. D i s t r i b u t i o n of g r a f t r e j e c t i o n scores of DBA/2 skin a l l o g r a f t s on +/+, W/+, Wv/+, and W/Wv mice; g r a f t s were excised on the eighth day a f t e r t r a n s p l a n t a t i o n . . 62 V. M o r t a l i t y data f o r DBA/2 skin a l l o g r a f t s on +/+, W/+, Wv/+, and W/Wv hosts /. . 63 VI. Graft r e j e c t i o n scores f o r g r a f t s of s k i n from +/+ male donors transplanted to +/+ and W/WV female hosts . 65 VII. Uptake of t r i t i a t e d thymidine by c e l l s of the p e r i -pheral blood of mice bearing s k i n a l l o g r a f t s . L a b e l l i n g done i n vivo 67 VIII. Uptake of t r i t i a t e d thymidine by c e l l s of the p e r i -pheral blood of mice bearing skin a l l o g r a f t s . L a b e l l i n g done ixi v i t r o 68 IX. Serum a g g l u t i n i n t i t e r s i n mice of d i f f e r e n t genotype a f t e r immunization with SRBC 71 X. A. Numbers of plaque-forming c e l l s produced by spleens of mice of d i f f e r e n t genotype four days a f t e r exposure to SRBC 74 B. Numbers of plaque-forming c e l l s produced by spleens of mice of different^genotype s i x days a f t e r exposure to SRBC 74 XI. Uptake of t r i t i a t e d thymidine (expressed as mean CPM) by spleen c e l l s of +/+ and W/Wv mice cultured with various concentrations of LPS and Con-A 76 v XII. Stimulation indices of spleen c e l l s from +/+ and W/W mice cultured with various concentrations of LPS and Con-A and l a b e l l e d with t r i t i a t e d thymidine 77 i x X Table Page XIII. Area (mm2) covered by p e r i t o n e a l c e l l s from mice of d i f f e r e n t genotype a f t e r 8 hours of migration from c a p i l l a r y tubes i n Mackaness-type chambers 79 XIV. Percentage of lymph node c e l l s adhering to glass cover s l i p s a f t e r 45 minutes incubation 80 XV. T o t a l and d i f f e r e n t i a l white blood c e l l counts of ungrafted +/+, W/+, Wv/+, and W/Wv mice 81 XVI. T o t a l and d i f f e r e n t i a l white blood c e l l counts of +/+, W/+, Wv/+, and W/Wv mice bearing skin autografts or skin a l l o g r a f t s 82 XVII. A summary of major experiments concerning immune responses i n W mutant mice 84 LIST OF FIGURES Figure Page 1. a. Normal mouse skin b. Relationship between graft and host tissue . . . . 14 2. Construction of PFC plates 29 3. Mackaness-type chamber for c e l l migration studies. . . 33 4. Ring chambers for c e l l adhesion studies 35 5. Skin isografts - 39 6. Skin allografts in the healing-in phase 43 7. Skin allografts entering the acute rejection phase . . 45 8. Skin allografts undergoing acute rejection 47 9. Skin allografts undergoing acute rejection 47 10. Skin allografts in the later part of the acute rejection phase 50 11. Skin allografts entering the replacement phase . . . . 50 12. Skin allografts in the replacement phase 53 13. Skin allografts in the replacement phase 55 14. Uptake of t r i t i a t e d thymidine by cells of the peripheral blood of mice bearing skin allografts or skin autografts. Labelling done in vitro 69 15. Serum agglutunin titers of mice of different genotype after immunization with SRBC 72 16. A simplified and schematic representation of some of the events occurring during the course'of a cellular immune response 90 xi LIST OF ABBREVIATIONS AFC antibody-forming c e l l ARC antigen-reactive c e l l B - c e l l thymus-independent c e l l BSA bovine serum albumen BSS balanced s a l t s o l u t i o n cAMP c y c l i c adenosine monophosphate Ci/mM Curies per mil l i m o l e Con-A Concanavalin-A CPM counts per minute DPM di s i n t e g r a t i o n s per minute ECF e o s i n o p h i l chemotactic f a c t o r H h i s t o c o m p a t i b i l i t y IgG immunoglobulin G IgM immunoglobulin M l r immune response LAF lymphocyte a c t i v a t i o n f a c t o r LPS lipopolysaccharide LT lymphotoxin MIF macrophage i n h i b i t i o n f a c t o r MST median s u r v i v a l time PFC plaque-forming c e l l PHA phy tohemagglutinin POPOP 1,4-Bis-[2-(5-phenyloxazolyi)]-benzene x i i x i i i PPO 2,5-diphenyloxazole PWM pokeweed mitogen RPMI Roswell Park Memorial Institute (tissue culture medium) S.A. specific activity SRBC sheep red blood cells TCA trichloroacetic acid T-cell thymus-dependent c e l l TF transfer factor ACKNOWLEDGEMENT I should l i k e to thank Dr. A. B. Acton f o r suggesting the o r i g i n a l problem from which t h i s thesis has developed, and f o r h i s advice and encouragement during the course of th i s study. I also wish to thank the members of my Thesis Committee, Dr, C. V. Finnegan, Dr. H. K. Kasinsky, and Dr. J . W. Thomas, f o r t h e i r comments and c r i t i c i s m s regarding my research and the preparation of t h i s t h e s i s . I am very g r a t e f u l to M. Douglas f o r ex c e l l e n t t e c h n i c a l a s s i s -tance at seve r a l stages i n t h i s work, and f o r the d r a f t i n g of the figures i n t h i s t h e s i s . In a d d i t i o n , I should l i k e to thank R. McMaster of the Department of Microbiology f o r h i s help with the techniques used i n the mitogen s t i m u l a t i o n experiments, and Dr. J . Levy, a l s o of the Department of Microbiology, f o r the use of c e r t a i n materials and f a c i l i -t i e s i n her laboratory. F i n a l l y , I wish to say a s p e c i a l thank you to my colleagues, i n p a r t i c u l a r Pat Collen, C. Boogaard, and E. S l a v i n s k i , with whom I have shared many hours of discussion and who have been a continual source of encouragement to me. During the course of t h i s study, I have been supported by a bursary and a scholarship from the National Research Council of Canada. x i v INTRODUCTION A mutation at the W locus i n mice was f i r s t recognized by i t s e f f e c t on coat colour and pigment pattern (Durham, 1908). The mutation, however, when homozygous i s also responsible for a severe macrocytic anemia and f o r s t e r i l i t y . Mice demonstrating these three seemingly unrelated e f f e c t s have proven p a r t i c u l a r l y s u i t a b l e f or a v a r i e t y of studies i n c e l l and developmental biology: experiments i n v o l v i n g pro-spective pigment c e l l s have l e d to a greater understanding of the genetic c o n t r o l of spotting patterns; studies of the anemia have y i e l d e d i n f o r -mation r e l a t i n g to the processes and c o n t r o l of hematopoiesis; and i n v e s t i g a t i o n s of the s t e r i l i t y of these mice have helped to e s t a b l i s h the extra-gonadal o r i g i n of p r i m o r d i a l germ c e l l s . In a d d i t i o n , c e r t a i n observations suggest that the e f f e c t s of t h i s p l e i o t r o p i c gene might include an e f f e c t on lymphocytes and immune responses. I have under-taken the i n v e s t i g a t i o n s described i n t h i s thesis i n the hope of demon-s t r a t i n g that the W mutant might prove as i n t e r e s t i n g and u s e f u l i n immunological studies as i t has i n developmental studies. In the presence of c e r t a i n modifying genes, the e f f e c t of the W mutation on pigmentation i s dominant, and h i s t o r i c a l l y t h i s gene has been c a l l e d "dominant s p o t t i n g " (Gruneberg, 1952). More commonly, W i s present with modifying genes which cause i t to behave as a semi-dominant spotting gene. A number of a l l e l e s e x i s t at the W locus, W, W , W , W , W , and W , each a f f e c t i n g pigmentation patterns i n 2 some way (Green, 1966). Mice carrying any one of these alleles exhibit variable white spotting and sometimes a slight dilution of colour as well. Mice carrying any two W alleles are, except for their eyes and occasionally the tips of their ears, entirely white. Such mice are also s t e r i l e and exhibit a severe anemia which is usually lethal within a few days of birth. Any W a l l e l e in combination with the WV a l l e l e , however, produces a less severe anemia and the animal survives. Studies of the W locus have, therefore, u t i l i z e d either WV/WV or W/WV mice almost exclusively. Numerous genes affecting pigmentation have been identified in mice, but those responsible for the various white spotting phenotypes have particularly interested developmental biologists. Spotting i s etiologically different from such colour anomalies as albinism. In albino animals, melanocytes are distributed normally in the tissues and are present in normal numbers, but are genetically incapable of producing tyrosinase, and hence can not produce pigment. In contrast, spotting genes result in a complete absence of melanocytes from the spotted area (Silvers, 1956). The spotting genes have been shown to affect pigment c e l l de-velopment via two routes, either directly, via the neural crest from which the pigment cells arise, or indirectly, via the environment through which the pigment cells must travel or in which they differentiate (Markert and Silvers, 1956). Belted (bt) and Steel (SI), for example, cause certain tissue environments to be unsuitable for the differen-tiation or survival of melanoblasts of any genotype (Mayer and Maltby, 3 1964; Mayer and Green, 1968). Dominant spotting (W), on the other hand, acts via the neural crest: some developmental defect caused by this gene precludes the production or differentiation of tnelano-blasts (Mayer and Green, 1968). Experiments suggest that in W embryos melanoblasts may be produced in smaller numbers than normal, and with a decreased a b i l i t y to survive or differentiate (Mayer, 1970). v The s t e r i l i t y of W/W mice i s associated with a profound defect in the proliferation of primordial germ cells (Mintz and Russell, 1957). In normal embryos, primordial germ cells appear in the yolk sac epi-thelium on the eighth day of development, and during the next four days migrate along the dorsal gut mesentery towards the genital ridges, proliferating markedly as they travel. In W/WV embryos, primordial germ cells are present in normal numbers at eight days, but proliferate very l i t t l e after this. The cells are also retarded in migration, so that very few cells ever reach the genital ridges. The gonads of newborn W/WV mice are thus severely deficient in germ c e l l s , and the adult animals are s t e r i l e . Of a l l hereditary mouse anemias, the severe macrocytic disorder exhibited by W/WV mice has been the most extensively studied (Green, 1966). The W series anemia i s characterized by a reduction in erythro-cyte numbers and an increase in mean c e l l volume. The severity of the disease depends on which alleles of the gene are present: some com-binations are lethal, causing general anoxemia in a l l tissues and resulting in death in utero or within a few days of birth (Borghese, 1959). 4 v The anemia of W/W mice can be permanently cured by the trans-plantation of marrow from normal, coisogenic animals (Bernstein and Russell, 1959); the transplanted cells apparently outgrow the indigenous c e l l population and quickly repopulate the anemic host. The defect produced by the W gene, therefore, clearly resides within the mutant hematopoietic cells themselves, and is not mediated by humoral or toxic factors in the environment, or by the absence of normal hematopoietic stimuli. v Marrow from W/W mice, when transplanted to irradiated, coiso-genic +/+ recipients, i s severely deficient in i t s capacity to form macroscopic spleen colonies (McCulloch, Siminovitch, and T i l l , 1964). Histological studies, however, reveal that W/WV marrow is capable of forming numerous microscopic colonies in the spleens of irradiated recipients (Lewis et a l . , 1967). The colonies produced by W/WV marrow, as compared to those produced by +/+ marrow, are fewer in number, smaller in size, slower to increase in size, and largely myeloid rather than erythroid in composition. Nevertheless, that they are produced at a l l indicates that W/WV marrow does contain a good supply of cells capable of hematopoietic differentiation. The reduced size and slower growth rate of colonies derived from W/WV c e l l s , however, "demonstrates a definite restriction of proliferation at early stages of [hematopoietic cell] differentiation" (Lewis et a l . , 1967). The marrow of W/WV mice may contain nearly normal numbers of stem c e l l s , but the effect of the W gene is to make these cells less able to multiply and differen-tiate than normal c e l l s . 5 I t i s c l e a r that n e i t h e r the s t e r i l i t y nor the c h a r a c t e r i s t i c pigment defect of W mutant mice i s secondary to the anemia: the neural crest defect has been demonstrated i n v i t r o , where conditions for mutant and non-mutant c e l l s are i d e n t i c a l ; the s t e r i l i t y i s maximal and i r r e -v e r s i b l e by the twelfth day of embryonic l i f e , although no hematopoietic anomaly i s yet apparent (Mintz and R u s s e l l , 1957). Each defect appears to r e s u l t from some e f f e c t of the W gene on a p a r t i c u l a r type of c e l l , e i t h e r prospective pigment c e l l , p r i m o r d i a l germ c e l l , or hematopoietic stem c e l l . The exact nature of the gene e f f e c t on each of these c e l l types, however, i s unknown and i s very much a subject of speculation. Both Mayer and Green (1968) and Bennett e_t a l . (1968) have noted that the a f f e c t e d c e l l types share the c h a r a c t e r i s t i c of being migratory and p r o l i f e r a t i v e , and have observed that i t i s migration and p r o l i -f e r a t i o n that i s d e f i c i e n t i n each of these c e l l l i n e s i n the mutant mouse. They have suggested that something necessary for these pro-cesses i s missing or a l t e r e d by the W gene. I f indeed something necessary f o r migration and p r o l i f e r a t i o n i s a l t e r e d by the W gene, then other migratory and p r o l i f e r a t i v e c e l l s might be af f e c t e d , too. Such a c e l l i n the adult animal i s the lympho-cyte, a c e l l capable of very extensive migration and, under appropriate stimulation, marked transformation and p r o l i f e r a t i o n . To t e s t the hypothesis that the W gene i n t e r f e r e s with migration, a study was made v i n our laboratory to determine i f the mutant W/W lymphocyte was indeed les s capable of migration than the normal +/+ lymphocyte (Wong, 1969). Lymph node c e l l s from e i t h e r W/WV mice or from t h e i r +/+ l i t t e r m a t e s 6 were cultured on monolayers of isogenic kidney c e l l s . Migrating cells were followed with time-lapse cinemicrography and the rate of motility determined in tracings from the micrographs. +/+ lymphocytes on +/+ monolayers were found to move at an average rate of 10.8 u per minute, and W/WV lymphocytes on W/WV monolayers at an average rate of 8.7 u per minute. S t a t i s t i c a l analysis indicates that these rates are signi-ficantly different. That W/WV lymphocytes do move more slowly than do +/+ lymphocytes suggests that something necessary for migration may indeed be the primary target of the W gene. In addition to an effect on migration, the W gene may also v v affect proliferation in lymphocyte lines. Spleens from W /W mice have been found by plaque assay to produce one-third to one-half as many hemolysin-forming cells as do spleens from W /+ mice (Shearer and Cudkowicz, 1967). This defect could be explained in three ways: 1) the presence of fewer antibody c e l l precursors in WV/WV mice; 2) a decreased a b i l i t y of WV/WV antibody c e l l precursors to respond to the proliferative stimulus; or 3) a decreased proliferative potential among individual antibody c e l l precursors. Studies of hematopoiesis have suggested that normal numbers of stem cells are present i n W/WV mice (Lewis et a l . , 1967; Bennett e_t a l . , 1968). Normal numbers of antibody c e l l precursors are, therefore, also l i k e l y to be present. The defective hemolysin response of WV/WV mice is most l i k e l y to be explained by some defect in precursor c e l l proliferation, similar to the defect in proliferation exhibited by other hematopoietic stem c e l l s . The work of Wong (1969) and of Shearer and Cudkowicz (1967), then, is not at variance with the hypothesis that something necessary 7 fo r migration and p r o l i f e r a t i o n may be missing or a l t e r e d i n c e l l s of W mice. Leaving, f o r the time being, speculation as to the primary s i t e of action of the W gene, I wish to consider the s i g n i f i c a n c e of the e f f e c t of such a gene on lymphocytes. Lymphocytes are w e l l known to be the key c e l l s concerned with immunological reactions of a l l kinds, i n c l u d i n g h y p e r s e n s i t i v i t y reactions, antibody synthesis, and t i s s u e r e j e c t i o n phenomena, and they appear to be involved at a l l stages of these reactions, from the induction of the response to the f i n a l e f f e c t o r phase. Indeed, lymphocytes are so intimately involved i n a l l phases of immune responses that any interference with t h e i r a c t i v i t i e s i s manifested by profound changes i n the development of the various immune reactions. Many aspects of these immune reactions, however, p a r t i c u l a r l y of such complex phenomena as ti s s u e r e j e c t i o n , are incompletely under-stood. The existence of an apparently abnormal lymphocyte population i n W mice prompted the speculation that t h i s aberration might be r e f l e c t e d i n some a l t e r a t i o n of immune phenomena i n these mice, and that these mice might then present a us e f u l i n vivo system f o r the i n v e s t i g a t i o n of those phenomena. The lymphocytes of W mice appear on the average to migrate some-what more slowly than lymphocytes from normal mice. The defect i n migration i s a small one, but lymphocytes play such a c e n t r a l r o l e i n immune responses that any defect i n t h e i r a c t i v i t y , however small, might be markedly amplified over the course of an immune response: a small defect i n migration might i n fac t have a large e f f e c t on reactions i n v o l v i n g the migrating c e l l s . Slow-moving lymphocytes might, f o r 8 example, take longer to reach p e r i p h e r a l areas where i n t e r a c t i o n with antigen may occur, and they might take longer to return to centres of lymphoid tis s u e where transformation and p r o l i f e r a t i o n are thought to occur, thus prolonging the e n t i r e response to antigen. Conversely, lymphocytes that move more slowly than normal might have greater oppor-tunity f o r exposure to and i n t e r a c t i o n with antigen: a greater number of lymphocytes might become a c t i v a t e d and the response to antigen might thus be enhanced. Likewise, a defect i n the p r o l i f e r a t i o n of lymphocytes from W mice might be expected to have profound e f f e c t s on the course of immune responses i n these mice. Drugs which i n t e r f e r e with lymphocyte p r o l i f e r a t i o n are commonly used to suppress immune responses, and, indeed, the W gene does appear to suppress the response to SRBC antigens. S i m i l a r l y , the gene might a f f e c t cell-mediated immune responses such as t i s s u e r e j e c t i o n , perhaps delaying the r e j e c t i o n c r i s i s by i n h i -b i t i n g the production of lymphocytes s p e c i f i c f o r the foreign t i s s u e . The existence of an aberrant lymphocyte population i n an other-wise immunologically normal animal i s p o t e n t i a l l y of great value i n immunological studies. Much i s known about lymphocytes and t h e i r a c t i -v i t i e s , but exactly how these c e l l s p a r t i c i p a t e and behave at various stages of immune reactions i s not f u l l y understood. How lymphocytes e f f e c t the destruction of s o l i d t i s s u e g r a f t s , f o r example, i s not e n t i r e l y c l e a r . In vivo studies of immune phenomena such as t i s s u e r e j e c t i o n often involve such gross manipulations of the immune system as neonatal thymectomy, thoracic duct drainage, treatment with 9 immunosuppressant drugs, or even i r r a d i a t i o n , manipulations which have very broad e f f e c t s on immune responses, e s s e n t i a l l y e l i m i n a t i n g at l e a s t some categories of lymphocyte a c t i v i t y e n t i r e l y . These manipu-l a t i o n s provide a l i m i t e d amount of information about the functions of lymphocytes, demonstrating, for example, that c e r t a i n classes of lymphocytes and c e r t a i n processes are e s s e n t i a l for p a r t i c u l a r immune reactions, but i n d i c a t i n g l i t t l e about the a c t i v i t i e s of those lympho-cytes during the course of those reactions. Attempts have been made to study immune phenomena i n v i t r o — t h e mixed lymphocyte re a c t i o n , f o r example, i s considered an i n v i t r o model for the process of a l l o g r a f t r e j e c t i o n — b u t i n v i t r o models, i n e l i m i n a t i n g many of the variables imposed by the i n vivo s i t u a t i o n , are n e c e s s a r i l y l i m i t e d i n t h e i r representation of the i n vivo process, and may not accurately represent what a c t u a l l y happens i n vivo. I f , however, a s i n g l e aspect of the character or behaviour of lymphocytes could be a l t e r e d i n some way without a l t e r i n g the immunological background of the animal, then the precise c o n t r i b u t i o n of lymphocyte a c t i v i t y to such complex phenomena as ti s s u e r e j e c t i o n could be more r e a d i l y examined i n vivo. Comparing immunological processes i n animals possessing an aberrant lymphocyte population with those processes i n animals possessing normal lymphocytes could reveal differences which might i n d i c a t e j u s t what the s i g n i f i c a n t events i n those processes are. Studies of abnormal reactions often lead, in. ways we can not p r e d i c t , to a b e t t e r understanding of normal reactions. The W mutant, then, appears eminently worthy of i n v e s t i g a t i o n . Experiments suggest a defect i n migration and p r o l i f e r a t i o n of 10 lymphocytes i n these mice, and i f these defects could be shown to have s i g n i f i c a n t e f f e c t s on immune phenomena, W mice might prove p a r t i c u l a r l y u s eful i n the furth e r i n v e s t i g a t i o n of those phenomena. I propose, therefore, to examine some aspects of both c e l l u l a r and humoral immune responses i n W mice to determine what e f f e c t , i f any, the W gene exerts on these reactions. In ad d i t i o n , I s h a l l attempt to consider the immuno-l o g i c a l e f f e c t s of th i s locus i n terms of a hypothesis f o r the primary s i t e of action of the W gene. MATERIALS AND METHODS I. MICE WBB6-F1 hybrid breeders of W/+ and WV/+ genotypes were obtained from the Jackson Laboratory i n August, 1970, and subsequently bred at the U n i v e r s i t y of B r i t i s h Columbia to produce hybrids of +/+, W/+, WV/+, and W/WV genotype. These mice, and t h e i r o f f s p r i n g produced by W/+ x WV/+ b r o t h e r - s i s t e r breeding, were used i n a l l experiments de-scribe d i n th i s t h e s i s . I n i t i a l studies, however, which required only +/+ mice, u t i l i z e d mice of the C57B1/6J s t r a i n . Mice were always three to f i v e months o l d before being s e l e c t e d f o r experiment. Mice of the DBA/2J s t r a i n served as ti s s u e donors i n a l l ex-periments i n v o l v i n g a l l o g r a f t s . Mice of t h i s s t r a i n d i f f e r from WBB6 mice at many l o c i , i n c l u d i n g H-2, and DBA ski n g r a f t s e l i c i t a strong r e j e c t i o n response i n WBB6 hosts. I I . SKIN GRAFT STUDIES The r e j e c t i o n of transplanted t i s s u e involves a complex c e l l -mediated immune response, a response i n which lymphocytes play a key r o l e . Any inte r f e r e n c e with normal lymphocyte movement, p r o l i f e r a t i o n , or function, as by severance of lymphatic drainage from the g r a f t s i t e , depletion of lymphocyte numbers, or i n h i b i t i o n of lymphocyte c e l l 11 12 d i v i s i o n cycles, i s manifested by a prolongation of the tissue r e j e c t i o n process and, h i s t o l o g i c a l l y , by a reduction i n the numbers of c e l l s i n f i l t r a t i n g the g r a f t . An i n h i b i t i o n of lymphocyte migration and p r o l i f e r a t i o n , such as has been suggested for the W gene, i s , therefore, also l i k e l y to be manifested by a prolongation of the r e j e c t i o n process. Skin has long been the t i s s u e of choice i n transplantation studies, l a r g e l y because of the ease with which i t can be transplanted and subsequently examined f o r s u r v i v a l . Skin also appears to be the most s e n s i t i v e of a l l tissues to h i s t o c o m p a t i b i l i t y d i f f e r e n c e s , and w i l l not survive i n d e f i n i t e l y unless donor and host are matched at a l l H l o c i . Any d i f f e r e n c e i n the a b i l i t y of two groups of animals to r e j e c t foreign t i s s u e i s l i k e l y to be manifest most r e a d i l y i n the response to s k i n a l l o g r a f t s . I chose therefore, to i n v e s t i g a t e the response of W/WV mice and t h e i r l i t t e r m a t e controls to skin a l l o g r a f t s . A preliminary experiment had suggested that g r a f t r e j e c t i o n by W/WV mice, rather than being slower than normal, might i n f a c t be some-what f a s t e r than normal. This r e s u l t was quite unexpected, and the following experiments were designed to determine i f indeed g r a f t re-j e c t i o n i n W/WV mice i s f a s t e r than i n +/+ mice, and i f t h i s d i f f e r e n c e i s detectable h i s t o l o g i c a l l y . A. Transplantation Technique The transplantation technique used throughout these studies i s b a s i c a l l y that described by Billingham and Medawar (1951). Mice which were to serve as skin g r a f t donors were k i l l e d by c e r v i c a l d i s l o c a t i o n , the l e f t side of the thorax shaved, and the skin 13 of t h i s region excised and placed i n Ringer's s o l u t i o n i n a P e t r i dish. Using a #11 s c a l p e l , the underside of the skin was scraped free of muscle, f a t , and blood vessels, leaving only the dermis and s u p e r f i c i a l e p i -dermis i n t a c t . This skin was cut into two or three pieces and each of these trimmed to a roughly oval shape, about 1 cm x 3/4 cm i n s i z e . The gra f t s were placed on f i l t e r paper moistened with Ringer's to await transplantation. Mice which were to receive s k i n g r a f t s were anaesthetized with .005-.0075 ml per gram body weight of Nembutal (sodium pentobarbitol, 60 mg/ml) d i l u t e d 1:4 with Ringer's and administered i n t r a p e r i t o n e a l l y . The l e f t side of the thorax was shaved and sprayed with Aeroplast. To form the g r a f t bed, the epidermis, dermis, and some subcutaneous f a t was c a r e f u l l y trimmed away from a region approximately 1 cm x 3/4 cm i n s i z e . The blood vessels which supplied t h i s area formed a f i n e network across the g r a f t bed, and were l e f t i n t a c t . Figure 1 i l l u s -t rates normal mouse ski n and the r e l a t i o n s h i p between g r a f t and host t i s s u e . The g r a f t i t s e l f was always the same s i z e as or only s l i g h t l y smaller than the g r a f t bed, and both were drie d with gauze before the gra f t was put i n place. The g r a f t was held i n place with a covering of vaseline-impregnated gauze and a p l a s t e r bandage wrapped around the body of the mouse. B. Measurement of A l l o g r a f t S u r v i v a l The reaction to a s k i n g r a f t can be measured most simply by determining the s u r v i v a l time of the g r a f t on the host. A comparison 14 Figure 1 Normal mouse skin. The epidermis i s th i n and densely stained. Hair f o l l i c l e s (hf) and pilosebaceous glands (pg) penetrate the dermis x200. Relationship between g r a f t and host t i s s u e . Panniculus  adiposus and Panniculus carnosus are removed from the g r a f t but retained i n t a c t i n the g r a f t bed. 15 } E p i d e r m i s D e r m i s P a n n i c u l u s a d i p o s u s P a n n i c u l u s c a r n o s u s G R A F T H O S T 5 3 E p i d e r m i s D e r m i s P a n n i c u l u s a d i p o s u s P a n n i c u l u s c a r n o s u s 16 of g r a f t s u r v i v a l times between experimental animals and t h e i r controls i s most s e n s i t i v e at the time which corresponds to the LD^^ or median s u r v i v a l time (MST) (Billingham et a l . , 1954a). The MST can be deter-mined by applying s k i n g r a f t s to a number of animals, checking the grafts every day f o r signs of r e j e c t i o n , and recording the end point. S t a t i s t i c a l procedures can be applied to the s u r v i v a l curve to y i e l d an estimate of the MST. Determination of the MST, however, requires only that the end point or day of t o t a l r e j e c t i o n of the g r a f t be recorded; i t requires scoring the .graft simply dead or a l i v e . On the other hand, a procedure i n which the grafts can be graded and given a numerical score i n d i c a t i n g the extent of the r e j e c t i o n process i n each of them provides more i n -formation than a simple dead or a l i v e c l a s s i f i c a t i o n , and i s therefore a more s e n s i t i v e measure than median s u r v i v a l time. I therefore chose i n my experiments to make h i s t o l o g i c a l studies of skin a l l o g r a f t s , studies which would permit not only a determination of the MST, but which would also allow comparisons to be made between each experimental a l l o g r a f t and i t s c o n t r o l . C. Establishment of C r i t e r i a by Which to Measure A l l o g r a f t S u r v i v a l 1) Isograft Studies In order to determine which changes i n an a l l o g r a f t are asso-c i a t e d with the r e j e c t i o n process, those changes associated with the wounding of the skin or with the healing process must f i r s t be defined. Isografts are prepared and transplanted in the same manner as are a l l o -grafts, but heal in permanently. They are by definition identical with the tissue of the host animal, and w i l l not e l i c i t any rejection reaction. Any changes isografts undergo can therefore be attributed only to the trauma of the transplantation procedure or to the healing process i t s e l f . Ten DBA/2 mice, males and females 9-12 weeks old, were grafted with skin from littermates of like sex. Starting on the fourth day after transplantation and continuing through the tenth day, two or three mice were k i l l e d on alternate days and the grafts prepared for histological study. 2) Allograft Studies a) Determination of histological c r i t e r i a for assessing a l l o - graft survival A preliminary study of skin allografts on C57B1/6 mice was undertaken in order to determine the type and time course of histolo-gical changes occurring in skin grafts undergoing rejection, and to establish a standard set of c r i t e r i a for measuring the progress of graft destruction. Eighty C57B1/6 mice, males and females 9-12 weeks old, were grafted with skin from DBA/2 donors. Donor and host were always of like sex. Starting on the third day after transplantation and continuing u n t i l the fifteenth day, five or six mice were k i l l e d each day and the grafts prepared for histological study as described below. 18 b) Determination of MST of DBA/2 gr a f t s on C57B1/6 hosts A f t e r determining h i s t o l o g i c a l c r i t e r i a f or assessing stages of a l l o g r a f t r e j e c t i o n , the s l i d e s of gr a f t t i s s u e were r e l a b e l l e d with code numbers, reordered randomly, and stored f o r several months. They were then re-examined using the es t a b l i s h e d c r i t e r i a , and scored on a scale of 1 to 6 f o r degree of r e j e c t i o n . For determination of the MST, however, only a dead or a l i v e c l a s s i f i c a t i o n i s required. Thus a l l o g r a f t s r e c e i v i n g g r a f t r e j e c t i o n scores of 1, 2, 3, or 4 were lumped together and c l a s s i f i e d " a l i v e , " and those r e c e i v i n g scores of 5 or 6 were considered "dead." From these data, the MST of DBA/2 ski n g r a f t s on C57B1/6 mice was determined, using a s i m p l i f i e d method of evaluating dose-response e f f e c t s described by L i t c h f i e l d and Wilcoxon (1948). v D. A l l o g r a f t Rejection i n W/W Mice: Grafts Exchanged Across H-2 B a r r i e r s Upon completion of preliminary studies to e s t a b l i s h h i s t o l o -g i c a l c r i t e r i a by which a l l o g r a f t r e j e c t i o n might be measured, two v experiments were designed to compare s k i n g r a f t s u r v i v a l i n W/W mice with that i n t h e i r +/+,. W/+, and WV/+ l i t t e r m a t e s . Preliminary h i s t o l o g i c a l studies had i n d i c a t e d that DBA/2 ski n g r a f t s on +/+ mice underwent t h e i r most marked degenerative changes between the seventh and eighth days. Any differe n c e i n the r e j e c t i o n process between +/+ and W/W mice, therefore was l i k e l y to be most apparent h i s t o l o g i c a l l y at the eighth day. An experiment was designed i n which 34 W/WV mice and 56 of t h e i r +/+, W/+, and WV/+ li t t e r m a t e s were grafted with DBA/2 skin. These mice were a l l k i l l e d on the 19 eighth day a f t e r t r a n s p l a n t a t i o n , and the ski n g r a f t s f i x e d f o r h i s t o -l o g i c a l study as described below. The s l i d e s were l a b e l l e d with code numbers and scored f o r degree of r e j e c t i o n on a scale of 1 to 6, using the c r i t e r i a described i n a) above. The d i s t r i b u t i o n of the gr a f t r e j e c t i o n scores of +/+ mice v was compared with that of W/+ and W /+ mice. Also, the d i s t r i b u t i o n v # of the g r a f t r e j e c t i o n scores of +/+, W/+, and W /+ mice together was compared with that of W/WV mice. The Mann-Whitney U-test ( S i e g e l , 1956) was used to determine whether or not the samples had been drawn from the same population. A second experiment was designed to measure the MST of DBA/2 skin on the experimental mice. F o r t y - f i v e W/WV mice, each paired with one or two lit t e r m a t e s of +/+, W/+, or WV/+ genotype, received g r a f t s of DBA/2 skin . Beginning on the seventh day a f t e r g r a f t i n g and con-ti n u i n g through the twelfth day, a number of W/WV mice and t h e i r con-t r o l s were k i l l e d each day and the ski n g r a f t s removed f o r h i s t o l o g i c a l examination. A l l o g r a f t s were prepared f o r microscopy as described i n the se c t i o n on h i s t o l o g i c a l technique. S l i d e s were l a b e l l e d with code numbers and scored f o r degree of r e j e c t i o n using the h i s t o l o g i c a l c r i t e r i a e s t a b l i s h e d e a r l i e r . A l l o g r a f t s r e c e i v i n g scores of 5 or 6 were considered dead. Mo r t a l i t y data f o r DBA/2 ski n a l l o g r a f t s on +/+, W/+, WV/+, v and W/W mice were analyzed using the method of L i t c h f i e l d and Wilcoxon (1949). Dose-effect s t r a i g h t l i n e s f o r each sample were p l o t t e d on lo g a r i t h m i c - p r o b a b i l i t y paper and the LD s f ) or MST read from the graph. 20 The method allows determination of 95% confidence i n t e r v a l s f o r each MST and permits an estimation of the heterogeneity of the MSTs of two samples. The method also permits a comparison of the dose-effect l i n e s f o r p a r a l l e l i s m . E. A l l o g r a f t Rejection i n W/WV Mice: Grafts Exchanged Across Weak  Hi s t o c o m p a t i b i l i t y B a r r i e r s Any d i f f e r e n c e between +/+ and W/W mice i n speed of g r a f t r e -j e c t i o n might be more apparent i n a t e s t u t i l i z i n g a l l o g r a f t s which i n c i t e a slower and l e s s vigourous response than do H-2 incompatible g r a f t s . Skin transplanted across H-2 b a r r i e r s survives only f o r a short h e a l i n g - i n period, then i s quickly rejected. There i s no reason to b e l i e v e that the h e a l i n g - i n period should d i f f e r between the two groups of mice. The length of the acute r e j e c t i o n period, however, i s r e l a t e d to the a n t i g e n i c i t y of the g r a f t , to the speed of the r e a c t i o n w i t h i n the draining lymph node, and to the speed with which mononuclear c e l l s can i n f i l t r a t e the g r a f t and exert t h e i r destructive e f f e c t s . It may not be p o s s i b l e to shorten the s u r v i v a l time of f i r s t - s e t g r a f t s beyond a c e r t a i n minimum, say 8 to 10 days, determined by the h e a l i n g -i n period and by some r a t e - l i m i t i n g step i n the r e j e c t i o n reaction i t s e l f . A stronger g r a f t r e a c t i o n , then, could not be detected i n terms of MST. I f , on the other hand, g r a f t s u r v i v a l were prolonged beyond the minimum time required f o r the r e a c t i o n i t s e l f , then di f f e r e n c e s between ex p e r i -mental and c o n t r o l groups might be more apparent. Female mice w i l l r e j e c t s k i n g r a f t s from coisogenic males which carry a Y-linked h i s t o c o m p a t i b i l i t y antigen ( S i l v e r s , 1968). Rejection across this weak histocompatibility barrier takes several weeks, and may therefore provide a more sensitive system for measuring small d i f -ferences between graft rejection times in different groups of mice. Skin grafts from male +/+ mice were prepared as described earlier (p. 12) and transplanted to female +/+ or W/WV littermate hosts. Twenty-one +/+ mice and six W/WV mice received grafts. Grafts were removed 17 to 24 days after transplantation, prepared for histological analysis, and scored blindly for degree of rejection using the histological c r i t e r i a established earlier. F. Histological Preparation of Skin Grafts Mice were k i l l e d by cervical dislocation and the grafts, with a narrow border of host skin, were excised, fixed in Allen's B-15 (1 hour at 37°C), and subsequently stored in Bouin's fixative (Culling, 1963). Allen's B-15 is a superior nuclear fixative (Baker, 1958) and seemed to give generally more satisfactory results than Bouin's alone. Tissue was dehydrated, embedded in Paraplast, sectioned at 7 or 8 p, and stained with eosin and Ehrlich's haematoxylin according to procedures described i n Culling (1963). III. LYMPHOCYTE ACTIVATION STUDIES The allograft response involves the stimulation and proliferation of a population of circulating lymphocytes. The number of such activated cells in the peripheral blood, as measured by the uptake of radioactive 22 DNA precursors such as t r i t i a t e d thymidine, i s considered a r e l i a b l e i n d i c a t o r of the imminence of g r a f t r e j e c t i o n . The following experi-ments were designed to determine whether or not g r a f t r e j e c t i o n i n W/WV mice i s associated with a greater number of activat e d c e l l s i n the p e r i p h e r a l blood of these mice than i s the g r a f t r e j e c t i o n of +/+, W/+, or WV/+ controls. A. L a b e l l i n g i n vivo Ten W/WV mice, males and females, each paired with a +/+, W/+, or WV/+ l i t t e r m a t e of l i k e sex, were grafted with skin from DBA/2 donors. Four hours before the mice were to be k i l l e d on the seventh, eighth, nineth, and tenth days a f t e r transplantation, they were i n j e c t e d i n t r a -p e r i t o n e a l l y with 1 yC per gram body weight of t r i t i a t e d thymidine (S.A. 20 Ci/mM, ICN, C a l i f o r n i a ) . Mice were bled from the o r i b i t a l sinus with 50X c a p i l l a r y p i p e t t e s , and .05 ml from each mouse was pre-pared f o r s c i n t i l l a t i o n counting (p. 23). Five +/+, W/+, and WV/+ mice and two W/WV mice which had not been grafted were also sampled as untreated controls. B. L a b e l l i n g i n v i t r o Ten W/WV mice, male and female, each paired with a +/+ l i t t e r -mate of l i k e sex, received DBA/2 skin g r a f t s . Two a d d i t i o n a l p a i r s of W/WV and +/+ mice received autografts. On the fourth, eighth, and fourteenth days a f t e r transplantation, blood samples were taken from each of three p a i r s of a l l o g r a f t e d mice; the autografted p a i r s were sampled on days four and eight. Two W/Wv and four +/+ mice which had not been grafted were also sampled as untreated c o n t r o l s . Mice were bled from the o r i b i t a l sinus with 50 A c a p i l l a r y p i p e t t e s . Samples of .1 ml were placed i n 12 x 100 mm glass t e s t tubes containing 1 ml of t i s s u e culture medium and 1 uC t r i t i a t e d thymidine. T r i p l i c a t e cultures were established f o r each mouse. Tubes were closed with Para-f i l m and incubated f o r four hours at 37°C. At the end of the culture period, c e l l s were harvested f o r s c i n t i l l a t i o n counting. The t i s s u e culture medium used i n a l l experiments i n t h i s s e r i e s was Dulbecco's modification of Eagle's medium supplemented with 15% i n a c t i v a t e d horse serum, L-glutamine, p e n i c i l l i n , streptomycin, and heparin. T r i t i a t e d thymidine (S.A. 15 Ci/mM, s t e r i l e s o l u t i o n i n water) was obtained from ICN, C a l i f o r n i a . C. S c i n t i l l a t i o n Counting A f t e r four hours exposure to t r i t i a t e d thymidine e i t h e r i n vivo or i n v i t r o , samples of whole blood were prepared f o r l i q u i d s c i n -t i l l a t i o n counting. Samples o f blood which had been l a b e l l e d i n vivo were placed i n 12 x 100 mm glass t e s t tubes. In v i t r o cultures were centrifuged at 800 g f o r 10 minutes to sediment the c e l l s , and the supernatant was removed. To each sample was added .03 ml c o l d thymidine, .03 ml c a r r i e r (2% BSA i n 10% s a l i n e ) , and .09 ml sodium l a u r y l sulphate. Samples were p r e c i p i t a t e d with 3 ml cold 5% TCA and incubated at 4°C f o r 1/2 hour. The p r e c i p i t a t e was sedimented by spinning at 2,500 rpm i n a c l i n i c a l centrifuge for ten minutes; the supernatant was then 24 removed. The p e l l e t was resuspended and washed twice with 3 ml cold TCA and 3 ml cold absolute ethanol. A f t e r the l a s t wash, 1 ml NCS tis s u e s o l u b i l i z e r was added to the p e l l e t , and the tubes were sealed and incubated at 37°C overnight. The samples were then t r a n s f e r r e d to s c i n t i l l a t i o n v i a l s and the tubes rinsed three times with 1.5 ml s c i n t i l l a t i o n f l u i d . These washings were added to the samples i n the s c i n t i l l a t i o n v i a l s and the volume made up to 15 ml with s c i n t i l l a t i o n f l u i d , a toluene s c i n t i l l a t o r s o l u t i o n containing 6 g/1 PPO and .75 mg/1 POPOP. V i a l s were stored i n the dark at 6°C for 24 hours before counting i n a Nuclear Chicago Isocap 300 l q u i d s c i n t i l l a t i o n counter. Each sample was counted f o r three 40 minute i n t e r v a l s , and the counts averaged. The channels r a t i o method was used to determine counting e f f i c i e n c y , and r e s u l t s are expressed as di s i n t e g r a t i o n s per minute, IV. HUMORAL IMMUNE RESPONSES The humoral immune response involves the production of c i r c u -l a t i n g antibodies to c e r t a i n foreign antigens. Mice respond to an i n j e c t i o n of foreign red blood c e l l s by producing antibodies capable of aggluntinating the i n j e c t e d c e l l s , or, i n the presence of complement, of l y s i n g them. The production of hemagglutinins can be monitored by standard s e r i a l d i l u t i o n techniques which i n d i c a t e the serum t i t e r of c i r c u l a t i n g antibody. The production of hemolysins can be monitored most s e n s i t i v e l y by a technique which permits the number of c e l l s actu-a l l y producing antibodies to be estimated. The following experiments 25 were designed to determine whether any differ e n c e exists between +/+ and W/WV mice i n t h e i r a b i l i t y to produce agglutinating or l y t i c a n t i -bodies i n response to stimulation with sheep red blood c e l l s . A. Production of SRBC Agglutinins i n +/+ and W/Wv Mice Five W/WV mice and t h e i r +/+ lit t e r m a t e s were immunized with an i n t r a p e r i t o n e a l i n j e c t i o n of .1 ml of 20% SRBC i n Dulbecco's balanced s a l t s o l u t i o n . To induce a secondary response, mice were i n j e c t e d again 18 days a f t e r the immunizing dose. Mice were bled from the o r b i t a l sinus before immunization (day 0), again on the 4th day a f t e r immunization, and at regular i n t e r v a l s a f t e r t h i s . Mice were starved f o r 12 to 16 hours before bleeding. F i f t y m i c r o l i t e r s of blood were c o l l e c t e d at each sampling time i n micro-hematocrit c a p i l l a r y tubes. Blood was allowed to c l o t f o r one hour at room temperature and then f o r four hours at 4°C. The tubes were centrifuged at 15,000 g f o r f i v e minutes i n an Adams Micro-hematocrit centrifuge, then broken j u s t above the i n t e r f a c e between the serum and the c l o t , and the serum tr a n s f e r r e d to a clean capillary tube. Sera were then incubated f o r t h i r t y minutes i n a water bath at 60°C to i n -ac t i v a t e complement, and f i n a l l y stored at 4°C f o r 24 hours before t e s t i n g . Mouse sera were tested f o r the presence of SRBC agglutinins by the d i r e c t hemagglutination method. A micro-method using disposable p l a s t i c m i c r o t i t e r plates and m i c r o l i t e r volumes of the reactants was employed. Appropriate d i l u t i o n s (lOx, or lOOx and 150x) of each serum 26 sample in Dulbecco's BSS were prepared. 50 u l of BSS were placed in each well except the f i r s t in a series of eight wells. 50 M l of serum of the dilution to be tested were placed in the f i r s t well and in the second well. The contents of the second well were mixed by pipetting, and 50 y l transferred to the third well. In this manner, s e r i a l two-fold dilutions were made through seven wells. 50 y l from the seventh well were discarded, leaving the eighth well as a control with no serum. Two series of two-fold dilutions were prepared for each serum sample tested, one series starting with a serum dilution of lOOx and the other starting v/ith a serum dilution of 150x. 50 yl of .4% SRBC in BSS were then added to each well. The plates were incubated for 3 hours at room temperature and then overnight at 4°C. The degree of hemagglu-tination in each well was scored as ++, +, +-, or - according to the following c r i t e r i a : - smooth-edged button of cells in center of well +- granular film of cells surrounding small button or ring of cells + or ++ diffuse film of agglutinated cells covering bottom of well The t i t e r of the serum was taken to be the highest dilution giving definite agglutination, that i s , scoring ++ or +. B. Production of Plaque-forming Cells in +/+, W/+, WV/+, and W/WV Mice Mice of +/+, W/+, WV/+, and W/WV genotypes (four mice of each type) were immunized with an intraperitoneal injection of .1 ml of 20% SRBC in Dulbecco's balanced salt solution. Four or six days after 27 immunization a l l mice were k i l l e d by c e r v i c a l d i s l o c a t i o n and the spleens removed to Syracuse dishes containing 5 ml BSS. Each spleen was gently rubbed against a wire mesh and thereby broken apart to release clumps of c e l l s ; these clumps were then further dispersed by a s p i r a t i n g the suspension repeatedly through a tub e r c u l i n syringe f i t t e d with a 25 gauge needle. The suspension was then removed to a centrifuge tube and incubated i n an i c e bath f o r 10-15 minutes to allow any remaining clumps of c e l l s to s e t t l e out, a f t e r which the lower 1-2 ml of the suspension was removed with a Pasteur p i p e t t e . The remaining c e l l suspension was centrifuged at 1,200 g f o r 10 minutes, the supernatant removed, and the c e l l s resuspended i n 5 ml BSS. At t h i s stage, t o t a l c e l l counts were performed i n a hemocytometer, and a small volume of the suspension d i l u t e d to an appropriate concentration (about 2 x 10 6 cells/ml) f o r use i n the following t e s t s . To .35 ml of each spleen c e l l suspension was added .1 ml of a 10% s o l u t i o n of washed, packed SRBC and .05 ml of undiluted guinea p i g serum. A f t e r thorough mixing, .1 ml of th i s mixture was placed i n each of three PFC plates (see below). The plates were sealed with melted vaseline and incubated at 37°C f o r 1 1/2 hours. Three plates were prepared f o r each mouse tested. Clear areas or plaques were p l a i n l y v i s i b l e and e a s i l y counted when the plates were observed against a l i g h t . The PFC plates used i n the above tests have been described by Cunningham (1968a). Ordinary glass microscope s l i d e s were washed i n 70% a l c o h o l and dried. At each end of one s l i d e was placed a piece 28 of Scotch brand tape which c a r r i e d adhesive on e i t h e r side; each piece of tape was 1/2 inch wide and long enough to span the s l i d e (Figure 2a). A second s l i d e was then placed on top of the f i r s t , s l i g h t l y out of r e g i s t e r so that a narrow l i p was l e f t along the long edge of the s l i d e (Figure 2b). The chamber thus created between the two s l i d e s and the pieces of s t i c k y tape was approximately one c e l l deep, and had a volume of about .1 ml. A f t e r adding a suspension of spleen c e l l s , SRBC, and guinea p i g complement, the chamber could be sealed by coating each l i p with melted vas e l i n e . V. MITOGEN STIMULATION STUDIES The response of lymphocytes to antigens i s mimicked i n v i t r o by the response of these c e l l s to lipopolysaccharide (LPS) from E_. c o l i and to various plant l e c t i n s such as phytohemagglutinin (PHA), pokeweed mitogen (PWM) , and Concanavalin-A (Con-A). These substances induce lymphocyte transformation and p r o l i f e r a t i o n s i m i l a r to that seen i n other circumstances where lymphocytes are be l i e v e d to be responding to foreign antigens. D i f f e r e n t mitogens appear to a c t i v a t e d i f f e r e n t populations of lymphocytes. In mice, f o r example, LPS has been shown to stimulate DNA synthesis i n B c e l l s only, and Con-A to stimulate DNA synthesis i n T c e l l s only (Andersson et a l . , 19 72a). These mitogens, then, provide a means of s e l e c t i v e l y inducing DNA synthesis i n T - c e l l or B - c e l l populations; the magnitude of the response to the two mitogens permits an estimation of the r e l a t i v e numbers of responding T- and B-29 Scotch double-stick tape i s placed at e i t h e r end of a p l a i n microscope s l i d e . A second microscope s l i d e i s placed on top of the f i r s t , s l i g h t l y out of r e g i s t e r . The space between the two s l i d e s i s about .1 ml i n volume and can be f i l l e d by c a p i l l a r y ' ac t i o n . The chamber i s sealed by p a i n t i n g the l i p s with melted va s e l i n e . Construction of PFC p l a t e s . 30 c e l l s i n a mixed population. The following experiment was designed to t e s t the responses of +/+ and W/WV spleen c e l l s , which are a mixed population of T- and B - c e l l s , to the mitogens Con-A and LPS, and thereby to determine i f the r e l a t i v e numbers of responsive T- and B - c e l l s are the same i n the two groups of mice. The following procedures were c a r r i e d out under s t e r i l e con-d i t i o n s . Mice were k i l l e d by c e r v i c a l d i s l o c a t i o n and the spleens removed to Syracuse dishes containing 5 ml of Dulbecco's balanced s a l t s o l u t i o n . Using needle probes, the organs were teased apart to release clumps of c e l l s . These were dispersed by a s p i r a t i n g the suspension repeatedly i n and out of a tub e r c u l i n syringe f i t t e d with a 25 gauge needle. The suspension of s i n g l e c e l l s was then centrifuged f o r 10 minutes at 1,500 g, the supernatant removed, and the c e l l s resuspended i n 5 ml of RPMI containing a n t i b i o t i c s and 10% f e t a l c a l f serum. T o t a l c e l l counts were performed i n a hemocytometer and the c e l l suspension i n each tube adjusted to a concentration of 2.5 x 10 6 c e l l s per ml.. Mitogen stimulation tests were performed i n flat-bottomed d i s -posable p l a s t i c m i c r o t i t e r p l a t e s (Linbro ISFB 96-TC) a technique adapted from Hartzman et a l . (19 71) by Pearson (19 74). 20 ml of c e l l suspension were deli v e r e d by automatic p i p e t t e (Hamilton Co. //PB-600-1) in t o each w e l l . .05 ml of mitogen, e i t h e r Concanavalin-A or Lipopolysaccharide, at various concentrations, were added to the c e l l suspensions; .05 ml of RPMI was added to the c o n t r o l cultures. Four r e p l i c a t e s were prepared for each culture condition. The plates were incubated at 48, 72, or 31 96 hours at 37°C i n a C0 2-enriched atmosphere. 16 to 18 hours before harvesting, .05 ml (1 uCi) of t r i t i a t e d thymidine (S.A. 2Ci/mM, New England Nuclear) was added to each w e l l . C e l l s were harvested by a s p i r a t i n g the contents of each w e l l through a milti-sample micro-harvester which drew the c e l l s onto discs of glass f i b e r f i l t e r paper (Pearson, 1974). Each f i l t e r d i s c was washed with 10 ml of d i s t i l l e d water and 5 ml of acetone, placed i n a s c i n t i l l a t i o n v i a l , and drie d at 110°C f o r 10 minutes. 5 ml of s c i n -t i l l a t i o n f l u i d were added to each v i a l . The v i a l s were placed i n a Nuclear Chicago Isocap 300 s c i n t i l l a t i o n counter and allowed to e q u i l i -brate f o r 12 to 24 hours before counting. Each v i a l was counted f o r 10 minutes. VI. OTHER STUDIES A. Macrophage Migration Studies Macrophages are a c t i v e migratory c e l l s and might be expected to be a f f e c t e d by a gene which i n h i b i t s migration i n some c e l l l i n e s . The following experiment was designed to determine whether or not the W gene has any gross e f f e c t on the migratory rates of mouse macrophages. Mice were i n j e c t e d i n t r a p e r i t o n e a l l y with 3 ml s t e r i l e mineral 011 to induce p e r i t o n i t i s . Three days l a t e r , these mice were k i l l e d by c e r v i c a l d i s l o c a t i o n and i n j e c t e d i n t r a p e r i t o n e a l l y with 5 ml Dulbecco's balanced s a l t s o l u t i o n . As much f l u i d as possible was with-drawn from the body cav i t y , and an i n c i s i o n then made i n the body w a l l . 32 The body cavity was washed with another 3 ml BSS, and t h i s wash added to the f i r s t . The suspension of c e l l s thus obtained was centrifuged at 800 g for 10 minutes; the c e l l s were washed once with Dulbecco's medium and f i n a l l y resuspended i n 2 or 3 drops of the medium. 20 X c a p i l l a r y tubes (Drummond) were f i l l e d with the c e l l suspension and sealed at one end by heating i n a Bunsen flame. These tubes were cen-t r i f u g e d at 800 g f o r 10 minutes to pack the c e l l s into the sealed end. The tubes were then broken j u s t below the i n t e r f a c e between the packed c e l l s and the supernatant to y i e l d a culture tube 3-5 mm long and f i l l e d with packed c e l l s . These culture tubes were then placed i n Mackaness-type chambers (Figure 3). Two c o n t r o l and two experimental c u l t u r e tubes were placed i n each chamber and held i n place with dabs of s i l i c o n e grease. The chamber was then f i l l e d with Dulbecco's medium, sealed with p a r a f f i n , and incubated at 37°C f o r 8 hours. At the end of the incubation period, the chambers were placed on the stage of a microscope f i t t e d with a p r o j e c t i o n eyepiece. The shadows of the c u l -ture tubes wi t h i n the chamber and of the area covered by the migrating c e l l s were c l e a r l y projected on a small screen placed near the micro-scope and could be recorded by t r a c i n g the outlines of the shadows. The area covered by the migrating c e l l s was then measured with a p l a n i -meter. P e r i t o n e a l exudate c e l l s from seven +/+, three W/+, f i v e and ten W/WV mice were tested. B. Lymphocyte Adhesion Studies Mice were k i l l e d by c e r v i c a l d i s l o c a t i o n and the p o p l i t e a l lymph nodes removed to a Syracuse dish containing 3 ml Dulbecco's balanced s a l t s o l u t i o n . Using needle probes, the nodes were teased 33 This chamber contains two culture tubes held i n place with s i l i c o n e grease, and shows the area covered by c e l l s migrating from each tube. The chamber i s sealed with round cover s l i p s and f i l l e d with medium v i a a syringe i n s e r t e d through a small hole i n one side. This hole i s sealed with melted wax. Figure 3. Mackaness-type chamber f o r c e l l - m i g r a t i o n studies. 34 apart to release clumps of c e l l s ; the clumps were further broken up by aspirating the suspension repeatedly in and out of a tuberculin syringe f i t t e d with a 26 gauge needle. The suspension of single cells thus obtained was centrifuged at 800 g for 10 minutes to sediment the cel l s . The supernatant was removed and the c e l l pellet resuspended in 2 ml Dulbecco's medium supplemented with 15% horse serum, antibiotics, and L-glutamine. At this stage, total c e l l counts were performed in a hemocytometer and the c e l l concentration adjusted to approximately 6 x 10 5 cells per ml. Culture chambers were constructed by attaching glass rings (15 mm internal diameter, 5 mm in height) to ordinary glass microscope slides with silicone grease (Figure 4a). Two rings were attached to each slide. About 1 ml of c e l l suspension was placed in each chamber and the chamber sealed with silicone grease and glass cover slips which had been precleaned in 70% alcohol (Figure 4b). The chambers were then inverted (Figure 4c), and were incubated for 30 minutes at 37°C. At the end of the incubation period, the number of lymphocytes per unit area which had settled on each cover s l i p was determined using an inverted microscope. Using a 40x objective and a grid placed i n a lOx ocular, the number of cells i n twenty different f i e l d s , ten fields on a vertical axis and ten on a horizontal axis, were counted. The average count per f i e l d was calculated for each culture chamber, and was usually about 30 to 50 ce l l s . After determining the average number of cells per f i e l d that were resting on the cover s l i p s , the chambers were righted (Figure 4d). Cells which had not adhered to the glass 35 c. Glass rings are attached to ordinary microscope s l i d e s with s i l i c o n e grease spread round one rim of the r i n g . Each r i n g i s f i l l e d with c e l l suspension and sealed with a cover s l i p and s i l i c o n e grease. The chamber i s inverted. The c e l l s s e t t l e on the cover s l i p and can be counted with an invert e d microscope. The chamber i s righted. The c e l l s adhering to the cover glass can be counted using a regular micro-scope. *P| i • fif^T Figure 4. Ring chambers f o r c e l l adhesion studies 36 cover slips f e l l away, but those which had become attached could be counted using a regular microscope. Again, using a 40x objective and a grid placed in a lOx ocular, the number of cells in twenty different fields were counted, and the average count per f i e l d calculated for each culture chamber. The number of adherent cells i s expressed as a percentage of the number of cells originally resting on the glass cover s l i p . Lymph node cells from seven +/+ and seven W/WV mice were tested. VII. HAEMATOLOGICAL TECHNIQUES Total and differential white blood c e l l counts were performed on ungrafted adult mice and on mice bearing skin allografts or skin v * autografts. At least eight mice of each genotype, +/+, W/+, W /+, and v W/W were sampled in each category. Mice were bled from the orbital sinus using heparinized capillary tubes. Because the sampling process i t s e l f can affect later counts, each mouse was bled once only. Standard haematological techniques were used throughout: total counts were' performed using an improved Neubauer chamber; blood films were stained with Wright's blood stain. RESULTS I. SKIN GRAFT STUDIES A. H i s t o l o g i c a l Features of Skin Isograft Healing Except f o r minor, t r a n s i t o r y abnormalities, i s o g r a f t s r e t a i n the appearance of normal sk i n throughout the h e a l i n g - i n period. Epi-. dermal hyperplasia, dermal oedema, and round c e l l i n f i l t r a t i o n , though never severe, are maximal at about the s i x t h day a f t e r t r a n s p l a n t a t i o n . By the eighth day, i s o g r a f t s are e s s e n t i a l l y i n d i s t i n g u i s h a b l e from normal s k i n . The h i s t o l o g i c a l features of i s o g r a f t healing are sum-marized i n Table I and i l l u s t r a t e d i n Figure 5. The appearance of numerous f i b r o b l a s t s i n the g r a f t bed and at the edges of the i s o g r a f t by the fourth day a f t e r t r a n s p l a n t a t i o n marks the formation of granulation t i s s u e . A p r o l i f e r a t i o n of new c a p i l l a r i e s i n these areas and i n the g r a f t dermis'indicates that the g r a f t i s healing i n . Very mild c e l l u l a r i n f i l t r a t i o n , mainly of poly-morphonuclear c e l l s , accompanies t h i s process. The epidermis at t h i s stage often separates from the dermis during h i s t o l o g i c a l preparation, but i s not h y p e r p l a s t i c or n e c r o t i c (Figure 5a). On the s i x t h day, c e l l u l a r i n f i l t r a t i o n of the g r a f t appears to reach a peak at a density only s l i g h t l y greater than at day four (Figure 5b). This i n f i l t r a t e consists of both mononuclear and poly-morphonuclear c e l l s , and i s confined mainly to the g r a f t bed and lower 37 38 TABLE I. H i s t o l o g i c a l features of s k i n i s o g r a f t healing. Days since transplan-t a t i o n Epithelium C e l l u l a r i n f i l t r a t i o n may be separated from very mild; mostly poly-dermis; no hyperplasia; morphonuclear no necrosis h y p e r p l a s t i c near edge of g r a f t appearance normal except f o r some hyperplasia at edge of g r a f t mild; greater proportion of mononuclear c e l l s than at day 4 very mild; mostly poly-morphonuclear 10 appearance normal l i t t l e or none 39 Figure 5. Skin i s o g r a f t s a. 4-day g r a f t x200. b. 6-day g r a f t x200. Only very mild hyperplasia and a l i g h t c e l l u l a r i n f i l t r a t i o n i n the g r a f t bed distinguishes s k i n i s o g r a f t s from normal ski n . There are no signs of e p i t h e l i a l necrosis at any stage. 41 part of the dermis. The epidermis at t h i s stage usually does not separ-ate from the dermis during h i s t o l o g i c a l preparation and i s normal i n appearance. Near the edges of the graf t i t may be somewhat h y p e r p l a s t i c , but necrosis never occurs. By the eighth day, only scattered polymorphonuclear c e l l s and the occasional mononuclear c e l l remain i n the g r a f t , and the epidermis looks e n t i r e l y normal. Only the density of f i b r o b l a s t s i n the granu-l a t i o n tissue of the g r a f t bed enables one to d i s t i n g u i s h the g r a f t i t s e l f from the surrounding host t i s s u e . By the tenth day, the g r a f t i s even more d i f f i c u l t to d i s t i n g u i s h from normal s k i n , and i s apparently permanently healed i n . B. H i s t o l o g i c a l Features of Skin A l l o g r a f t Rejection 1) General Observations The process of a l l o g r a f t r e j e c t i o n occurs i n three major phases: the h e a l i n g - i n phase, i n which granulation ti s s u e forms and the grafted t i s s u e becomes fi r m l y attached to the host; the acute r e j e c t i o n phase, i n which the g r a f t i s attacked and k i l l e d ; and the replacement phase, i n which the gr a f t i s sloughed and the wound repaired with a growth of new tiss u e from the host. a) The he a l i n g - i n phase For the f i r s t few days a f t e r t r a n s p l a n t a t i o n , a l l o g r a f t s r e t a i n the appearance of normal sk i n and are i n d i s t i n g u i s h a b l e from i s o g r a f t s . The epidermis may show mild hyperplasia, and a s l i g h t 42 i n f i l t r a t i o n of mononuclear c e l l s may occur i n the g r a f t bed (Figure 6a). Marked epidermal hyperplasia occurs only at the edge of the g r a f t (Figure 6b). A p r o l i f e r a t i o n of f i b r o b l a s t s i n the g r a f t bed and at the edges of the g r a f t marks the formation of granulation t i s s u e and the r e p a i r i n g of the wound (Figure 6c). The p r o l i f e r a t i o n of small blood vessels i s apparent i n the g r a f t bed by the fourth day, and q u i c k l y invades the lower layers of the g r a f t dermis (Figure 6d). By the f i f t h day or s i x t h day, the g r a f t appears f i r m l y attached to the host. b) The acute r e j e c t i o n phase Often as e a r l y as the fourth day a f t e r t r a n s p l a n t a t i o n , and almost i n v a r i a b l y by the f i f t h or s i x t h day, even before the hea l i n g process i s complete, a l l o g r a f t s begin to show degenerative changes which do not occur i n i s o g r a f t s . The mildly h y p e r p l a s t i c epidermis takes on a somewhat disorganized appearance and the basal c e l l s shrink and separate from each other (Figure 7). On the s i x t h day, the mono-nuclear c e l l i n f i l t r a t i o n of the g r a f t bed i s markedly greater than on the fourth day, and i s beginning to penetrate the dermis of the g r a f t (Figure 7). The blood vessels of the g r a f t bed are frequently l i n e d with mononuclear c e l l s perhaps about to migrate out i n t o the tissues (as i n Figure 6a). By the seventh day, the e n t i r e g r a f t epidermis may be markedly hy p e r p l a s t i c (Figures 8, 9) and scattered n e c r o t i c c e l l s can be found at the surface and i n the h a i r f o l l i c l e s . The epidermal c e l l s , which are normally somewhat b a s o p h i l i c , now s t a i n less densely, and some are even f a i n t l y e o s i n o p h i l i c . C e l l u l a r i n f i l t r a t i o n of the g r a f t i s denser and more extensive than on previous days. 43 Figure 6. Skin a l l o g r a f t s i n the h e a l i n g - i n phase. 3-day g r a f t x200. The epidermis i s th i n and densely-staining, and c e l l u l a r i n f i l t r a t i o n of the g r a f t bed i s l i g h t . e epidermis , , . g r a f t d dermis pa panniculus adiposus £j_ , , . , g r a f t bed pc panniculus carnosus 3-day g r a f t x400. Some epidermal hyperplasia i s apparent at the edge of the g r a f t as the wound i s repaired. e epidermis Epidermal hyperplasia and the formation of new connective ti s s u e (dermis) at the edge of a 4-day graf t x600. e epidermis ct connective tissue (dermis) Marked p r o l i f e r a t i o n of blood vessels i n the bed and dermis of a 4-day g r a f t x600. c c a p i l l a r y 45 Figure 7. Skin allografts entering the acute rejection phase. a. 4-day graft x400. b. 4-day graft x200. Epidermal hyperplasia i s moderate and cellular i n f i l t r a t i o n of the graft bed is light. Basal cells i n the epidermis may show distinct separation. Occasional pyknotic nuclei and vacuolated cells can be found. Degree of graft rejection: 2 Epith e l i a l survival: 90% 46 [ 47 Figure 8. Skin a l l o g r a f t s undergoing acute r e j e c t i o n . a. 5-day g r a f t x200. b. 6-day g r a f t x200. The epidermis of graf t s at t h i s stage shows more severe hyperplasia than at e a r l i e r stages; occasional c e l l s i n the h a i r f o l l i c l e s and sebaceous glands are pyknotic or vacuolated. The c e l l u l a r i n f i l t r a t e i s denser but s t i l l confined to the g r a f t bed and lower layers of the dermis. Degree of g r a f t r e j e c t i o n : 3 E p i t h e l i a l s u r v i v a l : greater than 50% Figure 9. Skin a l l o g r a f t s undergoing acute r e j e c t i o n . a. 6-day g r a f t x200. b. 7-day g r a f t x200. The g r a f t epidermis e x h i b i t s severe hyperplasia with many c e l l s con-t a i n i n g vacuoles or pyknotic n u c l e i (arrows). Hair f o l l i c l e s and sebaceous glands e x h i b i t widespread necrosis. The dense mononuclear c e l l i n f i l t r a t e at t h i s stage extends w e l l i n t o the dermis and may even reach the epidermis i n some g r a f t s . Degree of g r a f t r e j e c t i o n : 4 E p i t h e l i a l s u r v i v a l : l e s s than 50% 49 By the eighth and ninth days, e p i t h e l i a l necrosis i s widespread and often the h a i r f o l l i c l e s and sebaceous glands are l o s i n g t h e i r s t r u c t u r e . The epidermis i s severely h y p e r p l a s t i c and may be separated from the dermis by a dense l a y e r of invading c e l l s . Many e p i t h e l i a l c e l l s are f a i n t l y e o s i n o p h i l i c ; these are often vacuolated and may contain n u c l e i that are e i t h e r pyknotic or enlarged and v e s i c u l a r . C e l l u l a r i n f i l t r a t i o n of the g r a f t i s very dense, often extending i n t o the epidermis i t s e l f . By the tenth day a f t e r transplantation, many a l l o g r a f t s are e s s e n t i a l l y dead (Figure 10). The epidermis has l o s t i t s hyperplasia and i s very t h i n and e o s i n o p h i l i c . Although the o u t l i n e of epidermal and f o l l i c u l a r structures may p e r s i s t i n some cases, close i n s p e c t i o n reveals that they are h e a v i l y i n f i l t r a t e d with mononuclear c e l l s and that the e p i t h e l i a l elements themselves have d i s i n t e g r a t e d . Though s t i l l predominantly mononuclear, the c e l l u l a r i n f i l t r a t e i n the g r a f t seems to contain a somewhat greater proportion of polymorphonuclear leukocytes than on previous days. I n i t i a l changes i n the epidermal c e l l s of an a l l o g r a f t are not always or even usually associated with the density or proximity of the mononuclear c e l l i n f i l t r a t e . In some cases, mononuclear c e l l s are seen i n the lower s t r a t a of the epidermis before any marked de-gradative changes are apparent. Often, however, the epidermal c e l l s appear pale and n e c r o t i c even though the greater part of the c e l l u l a r i n f i l t r a t e i s confined to the lower layers of the dermis. In some cases, marked e p i t h e l i a l necrosis i s associated with only a very l i g h t 50 Figure 10. Skin allografts in the later part of the acute rejection phase. a. 9-day graft x200. b. 9-day graft x200. These allografts are essentially dead: hair f o l l i c l e s and sebaceous glands have lost most of their structure and are densely i n f i l t r a t e d with mononuclear and polymorphonuclear c e l l s . The epidermis, however, may s t i l l show severe hyperplasia and occasional nuclei retain their basophilia. Degree of graft rejection: 5 Epith e l i a l survival: less than 10% Figure 11. Skin allografts entering the replacement phase. a. 10-day graft x200. b. 12-day graft x200. A l l epidermal elements in these grafts, and even large portions of the dermis, have degenerated. Polymorphonuclear cells appear more abundant than in grafts at earlier stages. Degree of graft rejection: 6 Epithelial survival: 0% 51 52 c e l l i n f i l t r a t i o n that i s confined almost e n t i r e l y to the gr a f t bed. In other cases, a h y p e r p l a s t i c but non-necrotic epithelium i s asso-ci a t e d with an exceptionally dense and extensive c e l l u l a r i n f i l t r a t e . c) The replacement phase By the eleventh or twelfth day, v i r t u a l l y the e n t i r e g r a f t epidermis i s dead and i s being sloughed along with part of the g r a f t dermis (Figure 11). The c e l l u l a r i n f i l t r a t e i s s t i l l very dense, but polymorphonuclear leukocytes, p a r t i c u l a r l y e o s i n o p h i l s , and macrophages containing phagocytized material are more abundant than on e a r l i e r days (Figure 11). At the edges of the g r a f t , the host epidermis has begun to grow inwards, beneath the dead g r a f t and over the granulation ti s s u e that has formed (Figure 12a). In many cases, the deeper part of the gra f t dermis appears not to be sloughed, but to remain i n the g r a f t bed to be covered by th i s new growth of epithelium (Figure 12a). By the fourteenth or sixteenth day, host epidermis may cover much of the former g r a f t s i t e , and the c e l l u l a r i n f i l t r a t e i s beginning to decline. The new growth of epidermis appears to be invading the dermis at i n t e r -vals i n the formation of new h a i r f o l l i c l e s (Figures 12b, 13). 2) H i s t o l o g i c a l C r i t e r i a f o r Assessing Degree of A l l o g r a f t S u r v i v a l The h i s t o l o g i c a l changes occurring i n skin g r a f t s undergoing r e j e c t i o n can be used as c r i t e r i a for determining the stage of r e j e c t i o n of an i n d i v i d u a l g r a f t . Only those changes which occur during the acute r e j e c t i o n phase, however, are of relevance i n such a scheme. The condition of the epidermis at the surface of the gr a f t and i n the Figure 12. Skin a l l o g r a f t s i n the replacement phase. As the a l l o g r a f t i s sloughed o f f , the host epidermis grows out over the newly-formed dermis to r e p a i r the wound. The region bounded by the lower rectangle i s p i c t u r e d i n b below, and by the upper rectangle i n Figure 13. 16-day g r a f t x600. The new epidermis appears to be invading the dermis i n the formation of new h a i r f o l l i c l e s (arrows). 54 Old g r a f t bed A 5 5 Figure 13. Skin a l l o g r a f t i n the replacement phase. The rejected 16-day g r a f t i s separated e n t i r e l y from the host by a thin layer of new epidermis. The epidermis appears to be invading the dermis i n the formation of new h a i r f o l l i c l e s (arrows)(x600). 56 57 h a i r f o l l i c l e s , as demonstrated by the degree of hyperplasia and baso-p h i l i a , and by the frequency of n e c r o t i c c e l l s , i s the major i n d i c a t o r of the extent of g r a f t s u r v i v a l . The density and extent of the c e l l u l a r i n f i l t r a t e which accompanies g r a f t r e j e c t i o n i s also of some help i n assessing the magnitude of the reaction. Using these c r i t e r i a , s i x degrees of g r a f t r e j e c t i o n corresponding to successively lower percen-tages of s u r v i v i n g epithelium can be described (Table I I ) . Stage one describes those grafts which show no r e a l signs of abnormality and which are e s s e n t i a l l y i n d i s t i n g u i s h a b l e from normal sk i n . Some very mild epidermal hyperplasia may be present, but no signs of necrosis can be found. Few i f any mononuclear c e l l s have entered the grafted area. Stage two (Figure 7) describes those a l l o g r a f t s which show early but d e f i n i t e signs of abnormality. The epidermis i s moderately or severely h y p e r p l a s t i c and the c e l l s of the basal layer may show some separation. The epidermal c e l l s s t a i n l e s s densely than do the c e l l s of normal s k i n , and the c e l l membranes are well-defined. Very occasional pyknotic n u c l e i or vacuolated c e l l s can be found, but at l e a s t 90% of the epidermis appears healthy and v i a b l e . The g r a f t bed at t h i s stage shows only a very l i g h t i n f i l t r a t i o n of mononuclear c e l l s . Stage three a l l o g r a f t s (Figure 8) demonstrate somewhat more necrosis than do stage two a l l o g r a f t s . Pyknotic n u c l e i and vacuolated c e l l s are more frequent, hyperplasia may be more severe, and the c e l l u l a r i n f i l t r a t e i s denser and more extensive. At le a s t 50% of the epidermis appears v i a b l e . TABLE I I . H i s t o l o g i c a l features of s i x stages of skin a l l o g r a f t r e j e c t i o n i n normal mice. Degree of Time a f t e r % e p i t h e l i a l Swelling g r a f t transplan- s u r v i v a l and Staining Figure r e j e c t i o n t a t i o n hyperplasia character Necrosis C e l l u l a r i n f i l t r a t i o n Mono Poly 6 7 10 11 1 2 1-3 days 4- 5 days 5- 7 days 7-8 days 9-11 days 11+ days 100% 90% >50% <50% 10% 0% mild moderate severe severe severe b a s o p h i l i c l i g h t l y b a s o p h i l i c l i g h t l y b a s o p h i l i c mixed baso-p h i l i c and e o s i n o p h i l i c none rare +-+ scattered + or wide-spread e o s i n o p h i l i c wide- I I H spread e o s i n o p h i l i c complete +++ 59 Stage four a l l o g r a f t s (Figure 9) show rather severe damage, with less than 50% of the epithelium s u r v i v i n g . Epidermis, h a i r f o l l i -c l e s , and sebaceous glands r e t a i n t h e i r general s t r u c t u r e , but necrosis i s widespread. Many c e l l s are e o s i n o p h i l i c , and some are vacuolated; many contain e i t h e r enlarged, v e s i c u l a r n u c l e i or pyknotic n u c l e i . Epidermal hyperplasia i s severe, and the mononuclear c e l l i n f i l t r a t e may extend i n t o the epidermis i t s e l f . A l l o g r a f t s at stage f i v e are e s s e n t i a l l y dead, with le s s than 10% s u r v i v i n g epithelium (Figure 10). In many cases, the h a i r f o l l i c l e s and sebaceous glands have l o s t most of t h e i r s t r u c t u r e , and are densely i n f i l t r a t e d with mononuclear c e l l s . The epidermis i s severely hyper-p l a s t i c and i s often separated from the dermis by a dense l a y e r of i n -vading c e l l s . Most of the epidermal c e l l s are e o s i n o p h i l i c , although scattered n u c l e i r e t a i n some b a s o p h i l i a . The c e l l boundaries are often d i s t i n c t . A l l o g r a f t s at stage s i x are unquestionably e n t i r e l y dead (Figure 11). The epidermis has l o s t i t s h y p e r p l a s t i c appearance, and i s t h i n and e o s i n o p h i l i c ; i n d i v i d u a l c e l l s are d i f f i c u l t to d i s t i n g u i s h at a l l . In some cases, the o u t l i n e of epidermal and f o l l i c u l a r structures may p e r s i s t , but close inspection reveals that they are hea v i l y i n f i l t r a t e d with mononuclear and polymorphonuclear c e l l s . In other cases, epithe-l i a l elements are t o t a l l y degenerate and even the dermis of the g r a f t i s d i s i n t e g r a t i n g . A l l o g r a f t s at th i s stage are about to enter the replacement phase. 60 3) MST of DBA/2 Skin A l l o g r a f t s on C57B1/6 +/+ Mice The d i s t r i b u t i o n of gr a f t r e j e c t i o n scores for DBA/2 skin a l l o -g r a f t s on C57B1/6 +/+ r e c i p i e n t s i s shown i n Table I I I . S t a t i s t i c a l a nalysis of these data using the s i m p l i f i e d method of evaluating dose-response e f f e c t s described by L i t c h f i e l d and Wilcoxon (1948) reveals that the median s u r v i v a l time (MST) of these a l l o g r a f t i s 9.7 days. 95% confidence i n t e r v a l s f o r t h i s value are 8.6 days and 11.1 days. C. A l l o g r a f t Rejection i n W/WV Mice 1) On the EighthDay A f t e r Transplantation The d i s t r i b u t i o n of gr a f t r e j e c t i o n scores f o r DBA/2 ski n a l l o -grafts excised on the eighth day a f t e r t r a n s p l a n t a t i o n to 18 +/+ mice, 25 W/+ mice, 13 WV/+ mice, and 34 W/WV mice i s summarized i n Table IV. S t a t i s t i c a l analysis using the Mann-Whitney U-Test ( S i e g e l , 1956) shows that +/+ scores are not s i g n i f i c a n t l y d i f f e r e n t from W/+ and WV/+ scores; +/+, W/+, and WV/+ data can therefore be treated as samples from a s i n g l e population. Comparison of W/WV scores with +/+, W/+, and WV/+ scores, again using the Mann-Whitney U-Test, i n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e between the two samples (p = .0036); the two groups can not therefore be considered to be drawn from the same population. 2) MST of DBA/2 Skin A l l o g r a f t s on +/+, W/+, WV/+, and W/WV Hosts M o r t a l i t y data f o r DBA/2 ski n a l l o g r a f t s on 45 W/WV mice and 52 of t h e i r +/+, W/+, and WV/+ lit t e r m a t e s are summarized i n Table V. These data were analyzed using the s i m p l i f i e d method of evaluating 61 TABLE III. Graft rejection scores determined by histo-logical analysis of DBA/2 skin allografts on C57B1/6 +/+ hosts. Days a f t e r a transplantation Graft r e j e c t i o n scores 3 1 1 1 2 4 2 2 3 1 2 1 5 1 1 2 2 1 1 6 1 2 2 1 2 1 7 3 3 3 3 2 1 8 5 5 4 4 2 2 4 3 5 9 5 4 5 4 3 4 10 6 4 6 4 6 5 4 5 11 5 5 4 12 5 5 5 5 5 5 13 5 6 6 5 5 6 14 6 6 6 6 6 6 •k Each number in this column represents a single allograft on a single mouse. TABLE IV. Distribution of graft rejection scores of DBA/2 skin allografts on +/+, W/+, Wv/+, and W/Wv mice; grafts were excised on the eighth day after transplantation. Graft rejection score Number +/+ of mice W/+ obtaining wv/+ each score w/wv 1 3 4 1 3 2 1 10 2 3 3 5 6 6 7 4 6 3 2 10 * 5 3 1 2 9 * 6 0 1 0 2 n = 18 25 13 34 % mortality = 16.8 8.0 15.4 32.3 Graft rejection scores of 5 or 6 indicate that the graft is dead. 63 TABLE V. M o r t a l i t y data f or DBA/2 ski n a l l o g r a f t s on +/+, W / + , W v / + , and W / W v hosts. Days since M o r t a l i t y transplan-t a t i o n +/+ , W / + , w v / + w / w v # dead % # dead % # sampled mo r t a l i t y # sampled mo r t a l i t y 7 0/13 0 0/9 0 8 1/8 13 3/12 25 9 3/11 27 3/7 43 10 10/16 63 9/14 64 11 4/4 100 3/3 100 12 1/1 100 MST 9.7 days 9.3 days 95% confidence l i m i t s of MST 9.3-10.3 days 8.4-10.2 days 64 dose-effect experiments described by Li t c h f i e l d and Wilcoxon (1949). Dose-effect straight lines for each sample were plotted on logarithmic-probability paper. The LD 5 0 or MST of DBA/2 allografts on +/+, W/+, and WV/+ hosts given by this method is 9.7 days; 95% confidence limits for this value are 9.3 days and 10.3 days. The MST of DBA/2 grafts on W/WV hosts as read from the graph i s 9.3 days; 95% confidence limits for this value are 8.4 days and 10.2 days. Comparison of the two lines indicates that they do not deviate significantly from parallelism. Thus, the mortality rates of the two samples are not significantly different. A comparison of the MST values of the two samples indicates that these parameters are not significantly different. 3) Grafts Exchanged Across Weak Histocompatibility Barriers The graft rejection scores of skin from male +/+ mice trans-planted to female +/+ and W/WV hosts are shown in Table VI. The MST of allografts on +/+ hosts would appear to be about 20 or 22 days; un t i l day 20, more than 50% of the transplants on +/+ hosts are s t i l l alive (scoring less than 5). Although the number of W/WV hosts in this experiment is too small to allow definite conclusions to be drawn, v i t is interesting to note that none of the grafts to W/W mice showed any surviving epithelium, thus strongly suggesting a shorter MST for grafts on these mice. 65 TABLE VI. Graft r e j e c t i o n scores f o r grafts of ski n from +/+ male donors transplanted to +/+ and W/Wv female hosts. Days a f t e r transplan-t a t i o n Graft r e j e c t i o n +/+. scores w/wv 17 * 3,3,4,5 * 5 18 2,2,2,3,3 * * 5 ,5 20 * * * 4,5 ,6 ,6 22 * 3,4,6 * 6 24 * * * * 4,5 ,6 ,6 ,6 * * 6 ,6 Graft r e j e c t i o n scores of 5 or 6 i n d i c a t e that the g r a f t i s dead. 66 I I . LYMPHOCYTE ACTIVATION STUDIES A. L a b e l l i n g in vivo The uptake of t r i t i a t e d thymidine by c e l l s of the p e r i p h e r a l blood of +/+ and W/WV mice bearing no grafts or bearing s k i n a l l o g r a f t s i s shown i n Table VII. Results are expressed as d i s i n t e g r a t i o n s per minute per cul t u r e (.05 ml white blood) and as d i s i n t e g r a t i o n s per minute per 10 5 white blood c e l l s i n each cul t u r e . The mean DPM/105 white blood c e l l s of ungrafted +/+ mice i s lower than that of +/+ mice bearing a l l o g r a f t s . Unfortunately, samples of two ungrafted W/WV mice were l o s t , and comparisons between grafted and ungrafted mutant mice can not be made. S t a t i s t i c a l analysis using an unpaired t - t e s t i n d i c a t e s that on the eighth day a f t e r transplantation, mean DPM/105 white blood c e l l s from +/+ mice are s i g n i f i c a n t l y d i f f e r e n t from mean DPM/105 white blood c e l l s from W/Wv mice. Means at day seven, however, are not s i g n i f i c a n t l y d i f f e r e n t , and on days nine and ten the samples are too small f o r meaning-f u l comparisons to be made. I f a l l days are taken together, however, mean DPM/105 white blood c e l l s from +/+ mice are s i g n i f i c a n t l y d i f f e r e n t from mean DPM/105 white blood c e l l s from W/WV mice. B. L a b e l l i n g i n v i t r o The uptake of t r i t i a t e d thymidine by c e l l s of the p e r i p h e r a l blood of +/+ and W/Wv mice bearing s k i n a l l o g r a f t s i s shown i n Table VIII and summarized i n Figure 14. Results are expressed as d i s i n t e -grations per minute (DPM) per culture (.1 ml whole blood). Since only 67 TABLE VII. Uptake of t r i t i a t e d thymidine by c e l l s of the pe r i p h e r a l blood of mice bearing skin a l l o g r a f t s . L a b e l l i n g done i n vivo. Days after transplan-tation DPM/culture +/+ w/wv DPM/105 WBC +/+ w/wv * 7 1,395 1,639 698 815 1,319 1,051 222 629 1,110 291 1,072 1,562 509 577 * 8 1,092 360 909 1,020 266 455 1,198 774 1,391 384 1,121 2,613 436 409 1,012 256 1,139 1,036 397 516 9 1,125 1,108 534 820 10 1,159 1,143 731 543 1,169 322 no graft 1,165 223 986 155 1,082 261 913 142 969 258 249 127 356 108 462 158 * Student's Differences between +/+ and W/WV t-test, a = .025. mice are significant by 68 TABLE VIII. Uptake of t r i t i a t e d thymidine by c e l l s of the pe r i p h e r a l blood of mice bearing skin a l l o g r a f t s . L a b e l l i n g done i n v i t r o . Days a f t e r DPM /culture Mean No. of DPM/105 lymphocytes/culture lymphocytes +/+ W/WV +/+ W/W 367,700 298,200 transplan-t a t i o n +/+ w/wv 4 674 877 1,148 1,785 673 514 8 500 1,400 524 1,114 502 1,104 917 14 289 1,201 730 1,109 414 1,615 Day 8 autografts 62 95 157 370 No g r a f t s 451 171 713 305,600 305,300 434,700 595,800. 646,600 665,700 183.3 294.1 312.2 578.6 183.0 172.4 163.6 458.6 171.5 364.9 164.3 361.6 300.4 57.1 271.5 144.2 250.7 81.8 365.1 14.3 15.9 31.0 62.1 69.7 26.8 110.9 Minus background and DPMs "incorporated" at zero time (- 600 DPM). <n 5 0 0 -H o 4 0 0 sz CL H 3 0 0 9 2 0 0 Q. DL Q 1 0 0 a l l og r a f t s +/ + ? w/w" v~ "• autogra f t s 0 4 8 Days af ter Transplantation 1 4 Figure 14. Uptake of t r i t i a t e d thymidine by c e l l s of the peripheral blood of mice bearing skin a l l o g r a f t s or skin autografts. L a b e l l i n g done i n v i t r o . ON VO 70 ac t i v a t e d lymphocytes are known to incorporate t r i t i a t e d thymidine, however, r e s u l t s are also expressed as DPM per 10 5 lymphocytes. Since the number of lymphocytes may vary between c u l t u r e s , the conversion of DPM per culture to DPM per number of lymphocytes present i s a more s a t i s f a c t o r y representation of the r e s u l t s and provides a more accurate basis f o r comparison of c o n t r o l and experimental groups. DPM/105 lymphocytes from +/+ and W/WV mice bearing no g r a f t s or bearing autografts are c l e a r l y lower than DPM/105 lymphocytes from +/+ and W/WV mice bearing a l l o g r a f t s . The sample i s too small to allow a comparison between +/+ mice and W/WV mice bearing no g r a f t s , but among those bearing autografts analysis with an unpaired t - t e s t i n d i c a t e s no d i f f e r e n c e . Further s t a t i s t i c a l analysis with an unpaired t - t e s t i n d i c a t e s that on the fourth day a f t e r t r a n s p l a n t a t i o n , mean DPM/105 lymphocytes from +/+ mice are not d i f f e r e n t from mean DPM/105 lymphocytes from W/WV mice; however, on the eighth day and fourteenth day a f t e r trans-p l a n t a t i o n , mean DPM/105 lymphocytes from +/+ mice are s i g n i f i c a n t l y lower than mean DPM/105 lymphocytes from W/WV mice (a = .05). I I I . HUMORAL IMMUNE RESPONSES A. Production of SRBC Agglutinins i n +/+and W/WV Mice Serum t i t e r s of SRBC agglutinins produced by +/+ and W/WV mice over a period of ten weeks are shown i n Table IX, and are summarized i n Figure 15. The primary responses of +/+ and W/WV mice- are e s s e n t i a l l y TABLE IX. Serum a g g l u t i n i n t i t e r s i n mice of d i f f e r e n t genotype a f t e r immunization with SRBC. Serum ag g l u t i n i n t i t e r s a f t e r immunization with SRBC Mouse Geno- Days -> # type 0 4 6 8 1 0 1 4 2 0 2 4 2 8 3 2 3 6 4 2 5 2 7 2 1 +/+ 0 1 0 0 4 0 0 8 0 0 4 0 0 4 0 0 8 0 0 6 0 0 3 , 2 0 0 4 , 8 0 0 6 , 4 0 0 6 , 4 0 0 3 , 2 0 0 2 , 4 0 0 2 W / W v 0 2 0 0 4 0 0 4 0 0 4 0 0 2 0 0 2 0 0 6 0 0 1 , 2 0 0 1 , 6 0 0 8 0 0 3 0 0 3 +/+ 0 3 0 0 4 0 0 4 0 0 8 0 0 4 0 0 1 , 2 0 0 3 , 2 0 0 4 , 8 0 0 6 , 4 0 0 6 , 4 0 0 4 , 8 0 0 4 0 0 4 w / w v 0 3 0 0 6 0 0 6 0 0 8 0 0 4 0 0 4 0 0 1 , 2 0 0 3 , 2 0 0 2 , 4 0 0 3 , 2 0 0 2 , 4 0 0 1 , 2 0 0 6 0 0 5 +/+ 0 4 0 0 6 0 0 8 0 0 1 , 6 0 0 8 0 0 1 , 6 0 0 8 0 0 3 , 2 0 0 2 , 4 0 0 4 , 8 0 0 4 , 8 0 0 1 , 6 0 0 1 , 2 0 0 6 W / W v 0 4 0 0 6 0 0 8 0 0 8 0 0 4 0 0 8 0 0 4 0 0 1 , 6 0 0 1 , 6 0 0 3 , 2 0 0 4 , 8 0 0 3 , 2 0 0 8 0 0 7 +/+ 0 2 0 0 4 0 0 4 0 0 8 0 0 4 0 0 6 0 0 3 0 0 1 , 6 0 0 8 0 0 3 , 2 0 0 3 , 2 0 0 1 , 2 0 0 8 0 0 8 w / w v 2 0 2 0 0 3 0 0 4 0 0 8 0 0 4 0 0 6 0 0 6 0 0 1 , 6 0 0 8 0 0 3 , 2 0 0 4 , 8 0 0 1 , 2 0 0 8 0 0 9 +/+ 0 1 5 0 3 0 0 2 0 0 4 0 0 2 0 0 4 0 0 6 0 0 1 , 6 0 0 1 , 6 0 0 3 , 2 0 0 3 , 2 0 0 1 , 6 0 0 8 0 0 1 0 w / w v 0 2 0 0 3 0 0 4 0 0 8 0 0 4 0 0 4 0 0 4 0 0 4 0 0 4 0 0 8 0 0 8 0 0 8 0 0 8 0 0 I I I I I I I I 10 f 20 30 4 0 50 60 70 8 0 Ag Days a f ter Transplantat ion 15. Serum agglutinin tit e r s of mice of different genotype after immunization with SRBC. i d e n t i c a l . Most W/Wv mice, l i k e +/+ mice, are c l e a r l y capable of pro-ducing a prolonged secondary response to SRBC antigens but th i s response from the 32nd day onward, i s , on the average, s i g n i f i c a n t l y weaker than the secondary response produced by +/+ mice (Student's t - t e s t , a = .05). In some cases, the secondary response of W/W mice may be apparently normal, as i n mouse #6 and mouse #8. In other cases, the response i s somewhat less than normal, as i n mouse #2 and perhaps mouse #4. A secondary response seems to be lacking e n t i r e l y i n mouse #10, where a g g l u t i n i n t i t e r s never exceeded those of the primary response. B. Production of Plaque-forming C e l l s i n +/+, W/+, WV/+, and W/WV Mice The numbers of plaque-forming c e l l s (PFC) produced by mice of +/+, W/+, WV/+, and W/WV genotypes four days a f t e r immunization with SRBC are shown i n Table XA. Each number i n the body of the table represents the mean of t r i p l i c a t e samples from a s i n g l e mouse. Spleens from W/WV mice produce 1/4 the number of hemolysin-forming c e l l s as do spleens from +/+ mice. The number of PFC produced by spleens from W/+ and WV/+ mice i s intermediate between that produced by W/WV and +/+ mice. Analysis of these data using the K r u s k a l l - W a l l i s One-Way Analysis of Variance ( S i e g e l , 1956) indicates that the four samples are drawn from d i f f e r e n t populations. In normal mice, the peak PFC response to SRBC administered i n t r a p e r i t o n e a l l y occurs at four days (Wortis e_t a l . , 1966). At t h i s v time, as discussed above, spleens of W/W mice contain s i g n i f i c a n t l y fewer PFC than do spleens of normal mice. Tests were, however, also 74 TABLE XA. Numbers of plaque-forming c e l l s produced by spleens of mice of d i f f e r e n t genotype four days a f t e r exposure to SRBC. T r i a l # +/+ # PFC/10 5 Spleen W/+ C e l l s wv/+ w/wv 1 252 136 128 85 2 208 250 150 26 3 200 151 143 32 4 214 127 142 72 Mean 218 166 140 54 TABLE XB. Numbers of plaque-forming c e l l s produced by spleens of mice of d i f f e r e n t genotype s i x days a f t e r exposure to SRBC. T r i a l # # PFC/10 5 Spleen C e l l s +/+ w/wv 1 220 43 2 no data no data 75 performed on the s i x t h day a f t e r immunization i n consideration of the p o s s i b i l i t y that the k i n e t i c s of the PFC response i n W/WV mice might be a l t e r e d , with the peak occurring somewhat l a t e r . The a v a i l a b l e data are presented i n Table XB. There i s no i n d i c a t i o n that the peak PFC response of W/WV mice i s any higher s i x days a f t e r immunization than at four. IV. MITOGEN STIMULATION STUDIES The uptake of t r i t i a t e d thymidine by spleen c e l l s of +/+ and W/WV mice cultured with various concentrations of lipopolysaccharide (LPS) or Concanavalin-A (Con-A), and harvested a f t e r two, three, or four days of c u l t u r e i s expressed as mean counts per minute (CPM), and i s shown i n Table XI. Each number i n the body of the table represents the mean CPM of quadruplicate cultures of c e l l s from each of s i x mice. Using these data, s t i m u l a t i o n i n d i c e s (stimulation index = mean CPM of cultures with mitogens/mean CPM of RPMI cultures) were c a l c u l a t e d for each treatment and are shown i n Table XII. Both raw count means and the mean stimulation i n d i c e s of W/WV spleen c e l l s treated with LPS are always lower than those of +/+ spleen c e l l s , regardless of the concentration of mitogen used or the length of time i n culture. S t a t i s t i c a l analysis of the raw count means and of the stimulation indices using the Mann-Whitney U-test ( S i e g e l , 1956) confirms that +/+ and W/WV samples are drawn from d i f f e r e n t populations. 76 TABLE XI. Uptake of t r i t i a t e d thymidine (expressed as mean CPM) by spleen c e l l s of +/+ and w/wv mice cultured with various concentrations of LPS and Con-A. Mitogen concen-t r a t i o n 48 hour +/+ w/wv 72 +/+ hour w/wv 96 hour +/+ w/wv RPMI 2,430 2,782 907 1,337 1,071 1,834 LPS 5 44,285 40,430 23,514 13,948 11,493 2,166 LPS 20 47,721 37,165 27,677 16,162 13,453 2,891 LPS 40 42,374 38,919 19,758 16,167 8,717 8,371 Con-A 25 62,980 58,740 42,368 65,650 49,127 90,186 Con-A . 50 63,210 62,545 47,001 66,610 59,455 76,162 Con-A 1. 00 42,900 40,595 33,388 40,975 41,491 72,721 TABLE XII. Stimulation indices of spleen c e l l s from +/+ and W/Wv mice cultured with various concentrations of LPS and Con-A, and l a b e l l e d with t r i t i a t e d thymidine Mitogen 48 hour 72 hour 96 hour concen-t r a t i o n . +/+ w/wv +/+ w/wv +/+ w/wv LPS 5 18.2 14.5 25.9 10.4 10.7 1.2 LPS 20 19.6 13.4 30.5 12.1 12.6 1.6 LPS 40 17.4 14.0 21.8 12.1 8.1 4.6 Con-A .25 25.9 21.1 46.7 49.1 45.9 49.2 Con-A .50 26.0 22.5 51.8 49.8 55.5 41.5 Con-A 1.00 17.7 14.6 36.8 30.7 38.7 39.7 mean CPM of cultures with mitogens mean CPM of RPMI cultures 78 v Stimulation i n d i c e s of W/W spleen c e l l s treated with Con-A are approximately equal to stimulation indices of +/+ spleen c e l l s treated with Con-A, regardless of the concentration of mitogen used or the length of time i n cu l t u r e . S t a t i s t i c a l analysis using the Mann-Whitney U-test confirms that +/+ and W/WV samples are drawn from the same population. The mean raw counts of W/WV spleen c e l l s treated with Con-A, however, appear to be higher than those of +/+ spleen c e l l s a f t e r 72 or 96 hours of cul t u r e , and s t a t i s t i c a l analysis suggests v that +/+ and W/W samples at 96 hours are drawn from d i f f e r e n t popu-l a t i o n s . V. OTHER STUDIES A. Macrophage Migration Studies The area covered by p e r i t o n e a l exudate c e l l s migrating from c a p i l l a r y tubes i n Mackaness-type chambers was measured with a p r a n i -meter. The r e s u l t s are shown i n Table XIII. Each number i n the body of the table represents the mean of duplicate samples of c e l l s from a s i n g l e mouse. The area covered by migrating c e l l s v a ries markedly between mice, ranging from 17.0 to 22.5 mm2 f o r c e l l s from +/+ mice, with a mean of 18.7 mm2, and from 17.5 to 31.5 mm2 f o r c e l l s from W/+ and V i o W /+ mice, with a mean of 26.0 and 23.3 mnr r e s p e c t i v e l y . For c e l l s from W/WV mice, migration occurred over an area ranging from 11.5 to 42.5 mm2, with a mean of 21.7 mm2. There i s no apparent d i f f e r e n c e 79 between the four groups of mice; s t a t i s t i c a l analysis with the K r u s k a l l -Wallis One-way Analysis of Variance ( S i e g e l , 1956) confirms that the four samples are drawn from the same population. TABLE XIII. Area (mm2) covered by p e r i t o n e a l c e l l s from mice of d i f f e r e n t genotype a f t e r 8 hours of migration from c a p i l l a r y tubes i n Mackaness-type chambers. +/+ W/+ WV/+ W/WV 22.0 21.5 29.5 26.5 13.0 25.0 17.5 22.5 18.0 31.5 36.0 13.0 21.5 12.5 27.0 16.5 21.0 18.0 22.5 18.0 17.0 11.5 25.5 12.5 42.5 18.7 26.0 23.3 21.7 B. Lymphocyte Adhesion Studies The number of lymph node c e l l s adhering to glass cover s l i p s a f t e r 45 minutes of incubation i s expressed as a percentage of the number of c e l l s o r i g i n a l l y r e s t i n g on the cover s l i p . Results of tests of lymph node c e l l s from seven +/+ and seven W/WV mice are shown i n Table XIV. Each number i n the body of the table represents the mean 80 percentage of c e l l s adhering to cover s l i p s i n two d i f f e r e n t c u l t u r e chambers containing lymph node c e l l s from a s i n g l e mouse. There i s no apparent diffe r e n c e between the groups; analysis with the Mann-Whitney U-test confirms that the two samples are drawn from the same population. TABLE XIV. Percentage of lymph node c e l l s adhering to glass cover s l i p s a f t e r 45 minutes incubation. Genotype Percent Adherence Mean +/+ 28 30 33 40 46 46 52 . 39.3% w/wv 33 33 34 38 53 57 57 43.5% VI. HAEMATOLOGY A. Ungrafted Mice Haematological data f o r ungrafted mice, 12 to 16 weeks o l d , are shown i n Table XV. Mean white blood c e l l counts f o r female mice are c o n s i s t e n t l y lower than mean c e l l counts f o r ungrafted males, but the d i f f e r e n c e s are neither large nor s t a t i s t i c a l l y s i g n i f i c a n t . Some v a r i a t i o n e x i s t s i n t o t a l and d i f f e r e n t i a l counts between mice of d i f -ferent genotypes, but again the differences are not s i g n f i c a n t . 81 TABLE XV. T o t a l and d i f f e r e n t i a l white blood c e l l counts of ungrafted +/+, W/+, W /+, and W/W mice. Genotype T o t a l WBC/mm3 % Lymphocytes n +/+ 9 7,356 ± 2,227 87.9 ± 5 . 8 10 cr 7,944 ± 2,347 83.8 ± 10.6 11 W/+ 9 5,936 ± 1,319 80.3 ± 8 . 3 12 5 7,525 ± 3,048 71.3 ± 18.6 16 wv/+ 9 6,377 ± 1,954 88.5 ± 4.0 15 5 7,560 ± 2,787 84.7 ± 9 . 0 8 w/wv 9 6,727 ± 3,336 94.9 ± 1.2 10 5 6,880 ± 3,36 7 87.5 ± 10.0 11 B. Mice Bearing Skin Autografts or Skin A l l o g r a f t s Mice bearing s k i n autografts of eight days standing appear to have somewhat lower t o t a l white c e l l counts than do ungrafted mice (Table XVI), but the number of mice sampled here i s too small to permit meaningful comparisons to be made. To t a l white blood c e l l counts i n mice bearing s k i n a l l o g r a f t s are approximately h a l f those of ungrafted mice. D i f f e r e n t i a l counts are normal i n the early days a f t e r t r a n s p l a n t a t i o n (that i s , to day s i x ) , but i n l a t e r days (days seven to ten) these appear somewhat a l t e r e d , the mean percentage of lymphocytes decreasing by 10 to 15%. V a r i a b i l i t y i n d i f f e r e n t i a l counts of these grafted mice i s greater than that i n d i f f e r e n t i a l counts of ungrafted mice. There appear to be no differences i n any of these parameters between mice of d i f f e r e n t genotypes bearing a l l o g r a f t s . TABLE XVI. Total and differential white blood c e l l counts of +/+, W/+, WV/+, and V W/W mice bearing skin autografts or skin allografts. Days after transplantation n +/+, W/+, wv/+ Total WBC/mm3 % lymphocytes n w/wv Total WBC/mm3 % lymphocytes Allografts to day 6 10 4,562 ± 1,530 80.6 ± 10.5 10 3,823 ± 2,012 78.0 ± 11.4 days 7 to 10 31 4,710 ± 1,822 64.7 ± 19.3 31 4,431 ± 1,530 68.9 ± 18.7 day 14 7 5,656 ± 2,035 No data 5 4,424 ± 1,849 No data Autografts day 8 2 5,502 79.0 2 6,338 94.0 DISCUSSION The i n v e s t i g a t i o n s described i n t h i s thesis were undertaken i n the hope of c l a r i f y i n g the extent to which the W locus a f f e c t s immune responses. No new information concerning the e f f e c t s of the W mutation i n mice has been presented f o r some years, and the primary s i t e of action of t h i s gene has remained, u n t i l now, as much a mystery as ever. I f t h i s gene were found to a f f e c t immune responses, however, and i f the nature of t h i s e f f e c t could be determined, some new l i g h t might be shed on the mechanism of act i o n of t h i s enigmatic gene, and W mutant mice might thereby prove u s e f u l i n studies of immune reactions. In the discussion to follow, the experiments and data that have been presented here w i l l be i n t e r p r e t e d i n terms of what i s known about immune responses i n general, and w i l l then be examined f o r what they reveal about the act i o n of the W locus. A summary of the r e s u l t s of my major experiments concerning the immune responses of W mice i s presented i n Table XVII. I. TRANSPLANTATION IMMUNITY A. Genetics of Transplantation 1) C h a r a c t e r i s t i c s of Transplanted Tissue Tissues vary greatly i n t h e i r a b i l i t y to survive a f t e r homo-transplantation. The degree of c e l l u l a r i t y and v a s c u l a r i t y of the 83 TABLE XVII. A summary of major experiments concerning immune responses in W mutant mice. Experiment Results Statistical Test 1. Skin allografts examined histologically 8 days after transplantation. 2. MST of skin allografts exchanged across strong histocompatibility barriers. 3. MST of skin allografts exchanged across weak histo-compatibility barriers. 4. Uptake of t r i t i a t e d thy-midine by peripheral blood cells of mice bearing skin allografts. 5. Agglutinin production in response to immunization with SRBC Graft rejection scores of W/W mice are higher than those of +/+ mice. +/+: MST = 9.7 days W/WV: MST =9.3 days (no significant difference) +/+: MST = 20-22 days W/Wv: MST appears to be less than that fcr +/+ mice. 8 days after transplantation, counts in W/Wv cultures are higher than counts in +/+ cultures. 1° response: no difference between +/+ and W/Wv mice. 2° response: titers in W/Wv mice are higher than titers in +/+ mice. Mann-Whitney U-test p = .0036 Litchfield and Wilcoxon method for evaluating dose-effect experiments. Lit c h f i e l d and Wilcoxon method for evaluating dose-effect experiments. Student's t-test p = < .05 Student's t-test p = < .05 6. PFC production in response to immunization with SRBC. W/W mice produce fewer PFC than do +/+ mice. W/+ and Wv/+ mice produce PFC inter-mediate in number between those of +/+ and those of W/Wv mice. Mann-Whitney U-test p - .014 Kruskall-Wallis One-Way Analysis of Variance p = < .02 continued TABLE XVII. (Continued) Experiment Results S t a t i s t i c a l Test 7. Uptake of t r i t i a t e d thy- LPS: W/WV cultures take up fewer Mann Whitney U-test midine by spleen c e l l s stimu- counts and exhibit lower stimu- p = .05 l a t e d with LPS or Con-A. l a t i o n indices than do +/+ cultures. Con-A: W/Wv cultures take up more counts at 96 hours than do +/+ c u l -tures but do not exhibit higher stimulation i n d i c e s . cn Ul 86 t i s s u e , i t s m i t o t i c a c t i v i t y upon h e a l i n g - i n , the nature and abundance of i t s ground substance, and the q u a n t i t a t i v e expression of transplan-t a t i o n antigens on i t s c e l l surfaces may a l l a f f e c t s u r v i v a l of the g r a f t . A l l o g r a f t s of bone, endocrine glands, and c e r t a i n organs such as l i v e r , f o r example, may show prolonged or i n d e f i n i t e s u r v i v a l i f donor and host are matched at major h i s t o c o m p a t i b i l i t y l o c i . Skin, however, appears to be one of the most s e n s i t i v e of a l l tissues to h i s t o c o m p a t i b i l i t y d i f f e r e n c e s , and w i l l not survive i n d e f i n i t e l y unless donor and host are matched at a l l H l o c i . Skin g r a f t s , i n f a c t , seem to provide "an unduly p e s s i m i s t i c picture of the fate of a l l o g r a f t s i n general" (Billingham and S i l v e r s , 19 71, p. 91). This s e n s i t i v i t y , however, makes ski n g r a f t s i d e a l l y s u i t e d f o r i n v e s t i g a t i o n s where subtle e f f e c t s on the a l l o g r a f t response are to be detected. 2) H i s t o c o m p a t i b i l i t y Genes Tissue c o m p a t i b i l i t y i s governed by a number of genes which code f o r c e l l surface components ( S n e l l et a l . , 1973). These structures c o n s t i t u t e transplantation antigens which, i f foreign to the host, can i n c i t e immune responses. Permanent g r a f t acceptance occurs only i f a l l the transplantation antigens present i n the g r a f t are also present i n the host. The H-2 locus of the mouse i s a major h i s t o c o m p a t i b i l i t y complex that has recently been recognized as c o n s i s t i n g of a number of t i g h t l y r l i n k e d genes which c o n t r o l a v a r i e t y of immunological phenomena (Shreff-l e r and David, 19 75). At e i t h e r end of the H-2 region are two l o c i , H-2K and H-2D, which play the predominant r o l e i n determining 87 transplantation immunity i n mice. The a l l e l e s at these l o c i can be s e r o l o g i c a l l y defined by t h e i r a b i l i t y to engender and react with a d i v e r s i t y of antibodies; the K and D a l l e l e s appear to code for the polypeptide portions of two glycoprotein moieties which can be i s o l a t e d from c e l l membranes ( S h r e f f l e r and David, 1975). Strong r e j e c t i o n responses are e l i c i t e d i n s k i n g r a f t s exchanged between animals carrying d i f f e r e n t K and D a l l e l e s , i n d i c a t i n g that the s e r o l o g i c a l l y detectable products of these genes are probably the c l a s s i c h i s t o c o m p a t i b i l i t y antigens. The b i o l o g i c a l function of these genes and t h e i r products, however, i s unknown ( S h r e f f l e r and David, 1975). A number of minor H genes, i n c l u d i n g X- and Y-linked h i s t o -c o m p a t i b i l i t y f a c t o r s , play a l e s s e r r o l e i n t r a n s p l a n t a t i o n immunity. (Bailey, 1963; B a i l e y and Hoste, 1970; Stimpfling and Reichert, 1971). Grafts exchanged between animals d i f f e r i n g at minor l o c i may survive for prolonged periods before f i n a l l y being rejected. These weaker h i s t o -c o m p a t i b i l i t y genes appear to act cumulatively, so that g r a f t r e j e c t i o n i s quicker when donor and host d i f f e r at more than one minor locus. 3) Other Genes A f f e c t i n g Immune Responses The s e v e r i t y of the immune response e l i c i t e d by an a l l o g r a f t depends not only upon d i s p a r i t y between donor and host at H-2K and H-2D l o c i , but also upon the a c t i v i t y of other genes both i n the H-2 complex and elsewhere i n the genome. Most l r (immune response) genes so f a r described c o n t r o l the amount of antibody produced i n response to s p e c i f i c antigens (Gunther, 1973; Mozes and Shearer, 1972), but 88 genes c o n t r o l l i n g s u s c e p t i b i l i t y to thyroglobulin autoimmunity, tolerance induction to BSA, and the production of reagins have also been defined (Gunther, 1973). In a d d i t i o n , two l o c i r egulating a l l o g r a f t reaponsos have been reported: a dominant, H-2 l i n k e d gene has been shown to govern the response of female mice to the male H-Y antigen (Bailey and Hoste, 1970; Gasser and S i l v e r s , 1971), and a dominant, non-U-2 li n k e d gene appears to govern the r e j e c t i o n of a l l o g e n e i c bone marrow c e l l s i n mice (Cudkowicz, 19 71). Immune responses to most i f not a l l antigens are l i k e l y to be regulated by one or more I r genes. I r genes may act i n many d i f f e r e n t ways to control immune response They may, for example, a f f e c t antigen processing, antigen recognition, the number of s p e c i f i c a l l y reacting c e l l s , a c t i v a t i o n and p r o l i f e r a t i o n , immunoglobulin production, and so on (Gunther, 19 73). McDevitt (19 75) has reported that at l e a s t some I r genes are also responsible f o r con-t r o l l i n g T - c e l l - B - c e l l i n t e r a c t i o n . The functions of I r genes and t h e i r mechanisms of a c t i o n , however, are apparently complex and are only beginning to be in v e s t i g a t e d ; a b e t t e r understanding of I r gene a c t i v i t y seems fundamental to a b e t t e r understanding of immune processes i n general. B. Recognition and S e n s i t i z a t i o n 1) Antigen Recognition by Lymphocytes The i n i t i a l event i n the development of tran s p l a n t a t i o n Immunity i s antigen recognition, the event by which immunocompetent host c e l l o 89 f i r s t i n t e r a c t with foreign material (Figure 16). This process i s a function of antigen-sensitive small lymphocytes, and i s probably mediated by immunoglobulin receptors on the lymphocyte surface (Altman et a l . , 1973; Greaves, 1970; Diener and Langman, 1975). The nature of the i n t e r a c t i o n between antigen-sensitive c e l l s and the antigens of the g r a f t , however, and the precise mechanism by which antigen-reactive c e l l s are triggered, are not c l e a r (Diener and Langman, 19 75). 2) S e n s i t i z a t i o n The response of the lymphoid system to antigenic s t i m u l a t i o n has been w e l l documented (Andre et a l . , 1962a, 1962b; Andre-Schwartz, 1964; Scothorne and McGregor, 1955; Scothorne, 1957; P i e r c e , 1967). Within twenty-four hours a f t e r a l l o g r a f t t ransplantation, changes are detectable i n the draining lymph node, and by 72 hours a marked increase i n c e l l d i v i s i o n i s apparent (Devlin and Ramm, 19 71). By 4 days, c l u s t e r s of large p y r o n i n o p h i l i c c e l l s are present i n the node (Parrott and de Sousa, 1966) and a c t i v a t e d lymphoid c e l l s are appearing i n the lymph ( H a l l , 1967). By the s i x t h day a f t e r t r a n s p l a n t a t i o n , c e l l s capable of t r a n s f e r r i n g immunity are present i n s i g n i f i c a n t numbers i n the p e r i p h e r a l blood (Billingham e_t a l . , 1962). This process, i n which lymphoid c e l l s respond to antigen, i s the process of s e n s i t i z a t i o n (Figure 16). With regard to s k i n a l l o g r a f t s , a number of agents, i n c l u d i n g "passenger" leukocytes, epidermal c e l l s , f i b r o b l a s t s , and the vascular endothelium, have been suggested as the source of the s e n s i t i z i n g a n t i -gens . 90 ure 16. A s i m p l i f i e d and schematic representation of some of the events occurring during the course of a c e l l u l a r immune response. These are discussed i n d e t a i l i n the text. Recognition: Antigen-sensitive c e l l s i n t e r a c t with g r a f t antigens. How and where t h i s occurs i s not known. Small lymphocytes may enter the g r a f t , i n t e r a c t with g r a f t antigens, and return to lymph nodes. Macrophages may i n t e r a c t with g r a f t antigens and return to lymph nodes, perhaps carrying g r a f t antigens bound to t h e i r surfaces. Graft antigens may enter lymphatic vessels and t r a v e l to lymph nodes. S e n s i t i z a t i o n : Lymphoid c e l l s which have been s p e c i f i c a l l y a c t i -vated by g r a f t antigens "transform" into large, hyperbasophilic c e l l s . C e l l d i v i s i o n occurs. Activated c e l l s are released i n t o the lymph and eventually enter the blood stream. Graft Rejection: S p e c i f i c a l l y a c t i v a t e d lymphocytes enter the gr a f t and may, upon i n t e r a c t i o n with g r a f t antigens, release various lymphokines: MIF and ECF cause macrophages and eosinophils to accumulate i n the area; TF and LAF a c t i v a t e other lymphocytes which may then also release lymphokines; LT may exert a cytotoxic e f f e c t on g r a f t c e l l s . ECF - eo s i n o p h i l chemotactic f a c t o r LAF - lymphocyte a c t i v a t i o n f a c t o r LT - lymphotoxin MIF - macrophage i n h i b i t i o n f a c t o r TF - tr a n s f e r f a c t o r 91 Spec if ical ly Sensitized Lymphocytes Inter act w i t h G ra f t Antigens LT MIF (ECF TF.LAF Host Lymphatic Vesse' Afferent a rm of Ce l lu lar I mmune Respon Effector A rm of Immune Response Transformation of Smal Lymphocytes intd Specifically Sensitized Lymphocytes Sensit ized •VV , i Lymphocytes B l o o d J Enter B lood -j St ream Centra l A rm of Immune Response 92 The origin of the vasculature in skin grafts and whether or not i t is, antigenic has long been a subject of controversy. Although in orthotopic full-thickness skin grafts in rabbits and in heterotopic grafts of hamster cheek pouch skin, the original graft vessels appear to constitute the permanent vasculature of the graft (Lambert, 1971; Haller and Billingham, 1964), other reports indicate that in rats and mice the graft vessels do not become functional again; instead, new vessels from the host appear to grow in along the pre-existing graft vessels (Converse and Ballantyne, 1962; Converse e_t a l . , 1965; Zarem et a l . , 1967; Zarem, 1969). If blood vessels in these grafts are indeed of host origin, the vascular endothelium can not be the source of anti-genic stimulation. The small population of "passenger" leukocytes normally carried by an allogeneic skin graft, on the other hand, does appear to be en-t i r e l y sufficient to sensitize the host (Steinmuller, 1969; Steinmuller and Hart, 1971; Barker and Billingham, 1972). Allografts of pure epi-dermis, however, which are largely devoid of passenger leukocytes, are rejected almost as quickly as full-thickness skin grafts (Billingham and Sparrow, 1954). Epidermal c e l l s , therefore, probably play the major role in i n i t i a t i n g skin graft sensitivity, and passenger cells at most a minor role (Billingham, 19 71). In addition, the fibroblast population undoubtedly carries transplantation antigens, and may also contribute to graft antigenicity. Just where the i n i t i a l confrontation between lymphocyte and antigen takes place is another point of uncertainty. Sensitization 93 may occur p e r i p h e r a l l y as immunologically competent c e l l s from the blood move through the vessels or parenchyma of the gr a f t and are ex-posed to g r a f t antigens. A l t e r n a t i v e l y , s e n s i t i z a t i o n may occur cen-t r a l l y , with antigenic material draining or being c a r r i e d by lymph-borne c e l l s from the graf t to the l o c a l lymph node and there i n t e r -acting with lymphocytes (Medawar, 1958). Intact lymphatic drainage has been proven necessary f o r rapid s e n s i t i z a t i o n to take place (Lambert et a l . , 1965; Barker and B i l l i n g -ham, 1967, 1968). Thus, f i r s t - s e t s k i n grafts which have a normal blood supply but no lymphatic drainage are not r e a d i l y r e j e c t e d and f a i l to s e n s i t i z e the host. Lymph draining the s i t e of ski n a l l o g r a f t s contains much c e l l debris ( H a l l , 1967), and chemicals such as f l u o r o -dinitrobenzene appear to combine with soluble proteins at the s i t e of a p p l i c a t i o n , then enter the lymph and flow i n t o l o c a l lymph nodes ( H a l l and Smith, 19 71). Thus, antigenic material appears to pass down the lymphatics and s e n s i t i z e c e l l s within the lymph nodes. P e r i p h e r a l s e n s i t i z a t i o n , however, may indeed occur. Although alymphatic grafts e x h i b i t prolonged s u r v i v a l , they do eventually s e n s i -t i z e the host, po s s i b l y as lymphocytes move through the gr a f t and i n t e r -act with g r a f t antigens (Tilney and Gowans, 19 71; F u t r e l l and Myers, 1972; Ti l n e y and Ford, 1974). In each of these studies, however, the p o s s i b i l i t y that g r a f t antigens may have reached lymphoid t i s s u e v i a the blood stream can not be excluded. Whether or not pe r i p h e r a l s e n s i -t i z a t i o n a c t u a l l y can occur, therefore, remains i n doubt. C. The Rejection Process 1) Role of Lymphocytes Lymphocytes have long been suspected of functioning i n the destruction of tumours and s o l i d t i s s u e a l l o g r a f t s . Almost i n v a r i a b l y , r e j e c t i o n of foreign t i s s u e i s associated with the movement of mono-nuclear c e l l s into the g r a f t . That i t i s the small lymphocyte, however, which i s the key c e l l concerned with g r a f t r e j e c t i o n has been demon-str a t e d i n a v a r i e t y of ways. A l l o g r a f t r e j e c t i o n e i t h e r does not occur or i s very prolonged i n animals depleted of small lymphocytes. This has been shown i n ra t s for donor-host combinations d i f f e r i n g at weak h i s t o c o m p a t i b i l i t y l o c i (McGregor and Gowans, 1964; Roser and Ford, 1972), and i n mice (Deaton and Bach, 1970), a f t e r chronic drainage of lymphocytes from the th o r a c i c duct, or a f t e r treatment with lymphocytotoxic agents. X - i r r a d i a t i o n and treatment with drugs which suppress the production of lymphocytes also prolong a l l o g r a f t s u r v i v a l to a degree dependent on the s e v e r i t y of the treatment (see, for example, Brent, 1958). Congenitally athymic (nude) mice (Kindred, 1971) and neonatally thymectomised mice ( M i l l e r , 1961) are also severely d e f i c i e n t i n lymphocytes, and e x h i b i t depressed immune responses. C e l l t r a n s f e r experiments have also implicated the small lympho-cyte i n g r a f t r e j e c t i o n . Adoptive t r a n s f e r of s e n s i t i z e d lymph node c e l l s causes f i r s t - s e t skin a l l o g r a f t s to be rejected i n second-set time (Billingham e_t a l . , 1954a), and t r a n s f e r of normal, unsensitized lymph node c e l l s can bring about the r e j e c t i o n of long-standing g r a f t s 9 5 on tolerant animals (Billingham ex al., 1956). In a d d i t i o n , t h o r a c i c duct c e l l s and p e r i p h e r a l blood c e l l s are as capable of t r a n s f e r r i n g immunity as are lymph node c e l l s (Gowans e_t aJL. , 1961; Billingham et a l . , 1962). F i n a l l y , studies on graft-versus-host reactions (Billingham. and Brent, 1959; Gowans, 1962; Hildemann, 1964) have shown that small lymphocytes are the immunologically competent c e l l s responsible f o r the graft-versus-host syndrome. Exactly how small lymphocytes accomp-l i s h such phenomena as a l l o g r a f t r e j e c t i o n , however, i s not c l e a r . 2) The C e l l u l a r I n f i l t r a t e A diagnostic feature of a l l o g r a f t s undergoing r e j e c t i o n i s the accumulation within the g r a f t of large numbers of white blood c e l l s , and most p a r t i c u l a r l y of mononuclear c e l l s . I t has been generally assumed that t h i s c e l l u l a r i n f i l t r a t e i s the means by which g r a f t r e -j e c t i o n i s accomplished. As part of an extensive study of a l l o g r a f t responses, J. B. Murphy (1926) i n v e s t i g a t e d the e f f e c t of depletion or s t i m u l a t i o n of the lymphoid system on the r e j e c t i o n process, and concluded that the i n f i l t r a t i n g c e l l s "are the agent through which the [ r e j e c t i o n ] mechanism exerts i t s f o r c e . " A number of h i s t o l o g i c a l studies of a l l o g r a f t r e j e c t i o n , u t i -l i z i n g both l i g h t and e l e c t r o n microscopy, have attempted to charac-t e r i z e and i d e n t i f y these c e l l s . There has, however, been incomplete agreement as to the i d e n t i t y of the c e l l s i n the i n f i l t r a t e . T i t u s and Shorter (1962) report that n e u t r o p h i l s , other polymorphonuclear leukocytes, and h i s t i o c y t e s are the predominant c e l l type i n s k i n a l l o g r a f t s undergoing r e j e c t i o n , but A l l e g r a e_t al_. (1968) report that reticulum c e l l s and lymphocytes predominate. Weiner ejt a l . (1964) report 96 that mononuclear c e l l s bearing some u l t r a s t r u c t u r a l s i m i l a r i t i e s to both lymphocytes and monocytes are the predominant c e l l associated with a l l o -g r a f t r e j e c t i o n , and, f o r want of a more accurate name, they term these c e l l s g r a f t r e j e c t i o n c e l l s . Titus and Shorter, however, were working with mice, A l l e g r a ejt a l . with monkeys, and Weiner et. al_. with r a b b i t s ; species differences may account i n part f o r the lack of agreement among the observations of these workers; In ad d i t i o n , the i d e n t i f i c a t i o n of many hematogenous c e l l s by morphological c r i t e r i a i s at best d i f f i c u l t , and i s complicated by a lack of standard nomenclature. D i f f e r e n t obser-vers develop d i f f e r e n t c l a s s i f i c a t i o n schemes and r e s u l t s from d i f f e r e n t laboratores are not r e a d i l y comparable. A unique study of the composition of the c e l l u l a r i n f i l t r a t e has been performed by Collen (19 74) using the techniques of Jakobisiak et a l . (1971) to i s o l a t e enzymatically the c e l l s i n f i l t r a t i n g mouse skin a l l o g r a f t s . Collen finds that lymphocytes, n e u t r o p h i l s , and macro-phages make up the greater proportion of the i n f i l t r a t e ; among these c e l l s , she has i d e n t i f i e d three classes of lymphocytes: t y p i c a l small lymphocytes, t y p i c a l large lymphocytes, and transformed lymphocytes. Collen also i d e n t i f i e d a group of b a s o p h i l i c c e l l s which she be l i e v e d were probably also lymphoid i n nature but were distin g u i s h e d from other small and large lymphocytes by t h e i r hyper-basophilic cytoplasm. How these various c e l l s come to be l o c a l i z e d i n the gr a f t and what t h e i r s p e c i f i c functions are, however, i s uncertain. I t has been generally assumed that among the c e l l s which accu-mulate i n a l l o g r a f t s are s p e c i f i c a l l y a c t i v a t e d lymphocytes that were produced i n l o c a l lymph nodes and released i n t o the blood. These c e l l s are presumed to "home" in t o the a l l o g r a f t and destroy i t (Figure 16). Prendergast (1964) l a b e l l e d i n vi v o those c e l l s o r i g i n a t i n g from a lymph node draining the s i t e of an a l l o g r a f t , and found that many of them d i d l o c a l i z e i n the g r a f t bed. The l o c a l i z a t i o n , however, d i d not appear to be s p e c i f i c ; l a b e l l e d c e l l s entered g r a f t s from unrelated donors with equal f a c i l i t y . H a l l (1967) l a b e l l e d i n v i t r o lymphoid c e l l s c o l l e c t e d from the e f f e r e n t lymph of a lymph node dra i n i n g the s i t e of an a l l o g r a f t , then r e i n j e c t e d the c e l l s i n t o the c i r c u l a t i o n of the host animal. He found that two to three l a b e l l e d c e l l s out of every 1,000 i n j e c t e d entered the homograft, whereas almost none entered an autograft on the same animal. Labelled c e l l s that had o r i -ginated i n response to a b a c t e r i a l antigen, however, entered the -homo-gr a f t with equal f a c i l i t y . Lance and Cooper (1972), on the other hand, using a unique double l a b e l l i n g technique, found that small numbers of a c t i v a t e d c e l l s did appear to home s p e c i f i c a l l y i n t o the g r a f t which had stimulated t h e i r production. These workers are convinced that l o c a l i z a t i o n i s s p e c i f i c ; the negative r e s u l t s of H a l l and Prendergast they f e e l are due to the d i f f i c u l t y of l a b e l l i n g only the s p e c i f i c a l l y a c t i v a t e d c e l l s , which are probably very few i n number. Lab e l l e d c e l l s , even i n Lance and Cooper's experiments, formed only a small percentage of the c e l l u l a r i n f i l t r a t e associated with g r a f t r e j e c t i o n . T i l n e y and Ford (19 74) also found "some degree of s e l e c t i v e accumulation of s e n s i t i z e d lymphocytes i n s p e c i f i c a l l o g r a f t s of s k i n " but warned that "the d i f f e r e n c e s were small and should be cautiously i n t e r p r e t e d . " 98 3) E f f e c t o r Mechanisms i n A l l o g r a f t Rejection a) S p e c i f i c i t y of g r a f t r e j e c t i o n One of the most s t r i k i n g c h a r a c t e r i s t i c s of s k i n a l l o g r a f t r e j e c t i o n i s i t s remarkable s p e c i f i c i t y . An elegant experiment by Mintz and S i l v e r s (19 70) u t i l i z i n g allophenic mice has demonstrated the s e l e c t i v e destruction of i n d i v i d u a l foreign c e l l s w i t h i n the narrow confines of s i n g l e h a i r f o l l i c l e s . When skin from allophenic donors was transplanted to isogenic parental s t r a i n mice, only those melano-b l a s t s and h a i r f o l l i c l e c e l l s which c a r r i e d foreign H-2 s p e c i f i c i t i e s were destroyed; those c e l l s which were of the same H-2 s p e c i f i c i t y as the host survived. Mintz and S i l v e r s did note that i f the majority of c e l l s i n the g r a f t were fo r e i g n , then the e n t i r e g r a f t would even-t u a l l y be destroyed as a r e s u l t of n o n s p e c i f i c n e c r o s i s . I f the l a r g e r part of the g r a f t were accepted, however, then r e j e c t i o n of the foreign portion was complete wit h i n two weeks and no f u r t h e r necrosis occurred. Any mechanism proposed to explain the destruction of s k i n a l l o g r a f t s must be able to account f o r such s p e c i f i c i t y . A number of i n v i t r o models are b e l i e v e d to represent various stages of a l l o g r a f t r e j e c t i o n i n vivo, and have been studied f o r the clues they o f f e r to the i n vivo process. Studies of i n v i t r o cyto-t o x i c i t y have demonstrated that target c e l l s may be k i l l e d by s e v e r a l mechanisms. Those most l i k e l y to function i n vivo are contact-induced ce l l - m e d i a t e d - c y t o t o x i c i t y and antibody-dependent c y t o t o x i c i t y , both of which are s p e c i f i c cytotoxic mechanisms. The r o l e of lymphokines i n c y t o t o x i c mechanisms, however, must also be considered. 99 b) Contact-induced cell-mediated-cytotoxicity It has been generally assumed that specifically activated lymphocytes accumulate in allografts and exert a direct and specific cytotoxic effect either on the vasculature of the graft or on the graft epithelium i t s e l f . Lymphocytes from sensitized donors have been shown to be specifically cytotoxic for target cells carrying the sensitizing antigens (Govaerts, 1960; Rosenau and Moon, 1961; Wilson, 1963; Perl-mann and Holm, 1969). Such cells cultured in vitro with target c e l l monolayers are reported to cluster around the target c e l l s , causing them to cease DNA synthesis and to eventually round up, become detached, and die (Wilson, 1967; Ax et a l . , 1968). Berke (1972) reports that sensitized cells which are able to destroy tumour cells in vitro are also able to retard tumour growth in vivo, and Wagner et a l . (19 72) report that cells sensitized in. vitro are able to bring about the rejec-tion of tumour and skin allografts i n vivo. Contact-induced c e l l -mediated-cytotoxicity, therefore, as demonstrated i n vitro, i s thought to play a major role in the rejection of allografts in vivo. The work of Berke (1972) and Wagner et a l . (1972) demonstrates that the cells responsible for in vitro cytotoxicity are also functional in vivo; the precise mechanism by which they induce graft destruction, however, is not necessarily the same in both systems. Indeed, the death of skin grafts is not, in fact, readily ascribed to contact-induced cytotoxicity directed against the vascular endothelium or against the epithelial cells themselves. Vascular stasis has been reported to occur early in the rejection process and has often been 100 cited as the main cause of graft necrosis (Rolle, 1959; Waksman, 19 74). The vasculature of mouse skin grafts, however, is probably of host origin (p. 92) and therefore can not be the primary target of specific effector mechanisms. In addition, skin is remarkably resistant to ischemia and can survive for long periods in vitro or in the anterior chamber of the eye where no vascular supply exists (Medawar, 1948). Brent (1958) has noted that the time interval between vascular breakdown and epithelial destruction is too brief for ischemia to be the cause of graft deterioration. Neither can epithelial necrosis be explained by the direct cytotoxic action of specifically activated lymphoid cells i n contact with the cells of the graft. Epithelial necrosis is often seen well before the invading cells reach the upper layers of the graft, and before any contact is made between lymphoid cells and epithelium (Figure 9). In addition, only a few of the lymphocytes entering the graft appear to be specific for the graft (p. 9 7). The concept of direct, contact-induced cytotoxicity by specifically activated lymphocytes, therefore, although favoured by many as the primary mechanism respon-sible for allograft rejection, appears to be an oversimplification of the rejection process, and even, in the words of Billingham and Silvers (1971), "a complete misrepresentation" of the actual events. c) Role of lymphokines Sensitized lymphocytes upon interaction with specific anti-gen are known to release into the medium a number of biologically active factors called lymphokines (Granger and Williams, 1968). Some of the 101 lymphokines have been f u n c t i o n a l l y characterized by t h e i r a b i l i t y to i n h i b i t the migration of macrophages (MIF, macrophage i n h i b i t i o n f a c t o r ) , to exert a chemotactic influence on macrophages and eosinophils (MCF and ECF, macrophage and e o s i n o p h i l chemotactic f a c t o r s ) , to a c t u a l l y k i l l c e l l s (LT, a n o n s p e c i f i c lymphotoxin), and to stimulate normal lymphocytes i n v i t r o to transform and divide (LTF, lymphocyte trans-formation factor) (Dumonde et a l . , 1969; Granger, 19 72). S e n s i t i z e d lymphocytes are also able to produce a " t r a n s f e r " f a c t o r (TF) which i s capable of t r a n s f e r r i n g s p e c i f i c antigenic s e n s i t i v i t y , i n c l u d i n g homograft s e n s i t i v i t y , to normal lymphocytes (Lawrence, 1973). The a c t i v i t y of MIF, MCF, and ECF can r e a d i l y account f o r much of the c e l l u l a r i n f i l t r a t i o n associated with g r a f t r e j e c t i o n . Unsen-s i t i z e d host lymphocytes which accompany t h i s i n f i l t r a t e may be con-verted to a s p e c i f i c antigen-reactive state by TF, such that they then behave as s p e c i f i c a l l y s e n s i t i z e d c e l l s : upon i n t e r a c t i o n with antigen, they transform and release lymphokines. A very few s p e c i f i c a l l y sen-s i t i z e d lymphocytes, then, entering the g r a f t , i n t e r a c t i n g with g r a f t antigens, and r e l e a s i n g TF, could convert many uncommitted host lympho-cytes to antigen r e a c t i v e states such that they too would be able to function i n g r a f t destruction. Upon i n t e r a c t i o n with antigen, both s e n s i t i z e d lymphocytes and lymphocytes r e c r u i t e d by the action of TF elaborate lymphotoxin (LT), a lymphokine that has been shown to be n o n - s p e c i f i c a l l y cytotoxic for target c e l l s i n v i t r o (Granger and Williams, 1968, 1971; Walker and Lucas, 1973; Kramer and Granger, 1975). Walker and Lucas (1973) 102 and Kramer and Granger (19 75) have suggested that s p e c i f i c i n v i t r o c e l l - m e d i a t e d - c y t o t o x i c i t y i s also mediated by LT, and Granger (1969) has suggested that LT i s the p r i n c i p a l means by which s p e c i f i c g r a f t destruction i s accomplished i n vivo (Figure 16). The p r e c i s e mechanism of the c y t o t o x i c reaction, however, i s f a r from c l e a r . Henney e_t a l . (19 74) have reported that c e l l - m e d i a t e d - c y t o t o x i c i t y and mediator produc-t i o n can be experimentally d i s s o c i a t e d : that c y t o t o x i c i t y and the production of lymphokines represent independent cell-mediated immune functions. The r o l e of lymphotoxin i n c e l l - m e d i a t e d - c y t o t o x i c i t y i n v i t r o i s , therefore, s t i l l uncertain, and i t s r o l e i n vivo subject to much speculation. d) Role of antibody The antigens coded f o r by h i s t o c o m p a t i b i l i t y genes i n c i t e not only transplantation immunity but humoral immunity as w e l l . Although the formation of antibodies i s now generally regarded as a normal part of the a l l o g r a f t response, the r o l e of antibody i n g r a f t r e j e c t i o n i s not c l e a r . Depending upon the source and quantity of the sera, the species and s t r a i n s of animals involved, and the kinds of g r a f t s used, antibody may e i t h e r prolong or c u r t a i l g r a f t s u r v i v a l , or have no e f f e c t at a l l . A number of early experiments (reviewed by Stetson, 1963) seem to i n d i c a t e that humoral antibodies can b r i n g about r e j e c t i o n of skin g r a f t s , but Winn (19 70) i s s k e p t i c a l of these experiments because the observations have not been highly reproducible. Even when a n t i -bodies are s u c c e s s f u l i n causing r e j e c t i o n , the process seems r a r e l y to be acute and the g r a f t s p e r s i s t for s e v e r a l weeks before being 103 sloughed. Jooste et_ a l . (19 73) have recently reported that, in rats, skin grafts are susceptible to damage by humoral antibodies only during a short period about two weeks after transplantation. Healing-in grafts and long-standing grafts, they report, are not susceptible to damage by antibodies. Although humoral antibodies are not essential for allograft rejection, antibody-dependent cell-mediated ly s i s may s t i l l play a role in the rejection process. Target cells incubated in vitro with very low titers of antibody are lysed by lymphoid cells from normal donors (Perlmann and Holm, 1969). This l y s i s requires contact between lym-phoid c e l l and the target c e l l , and occurs with antibody dilutions too high to give rise to conventional complement-mediated l y s i s . Low titers of antibodies reactive against major histocompatibility antigens are undoubtedly formed in mice bearing skin allografts (Brazenor and Davies, 1973; Staines et a l . , 1974), and serum from such mice has been shown to induce cell-mediated cytotoxicity i n vitro (Degiovanni and Lejeune, 1973). Whether or not antibody-dependent cell-mediated lysis actually plays a role i n skin allograft rejection, however, is very uncertain. e) Conclusion Clearly, the mechanism by which tissue allografts are de-stroyed is a complex and as yet poorly understood process; no hypo-thesis has been put forth which satisfactorily explains a l l the obser-vations. Sensitized lymphocytes, lymphokines, and specific antibody are able to induce cytotoxicity in vitro, but the role of these various 104 effectors in vivo i s not at a l l clear. Specific skin graft rejection may very well be a result of the combined activity of each of these specific mechanisms, and of other non-specific tissue-damaging processes as well. v D. Skin Allograft Rejection in W/W Mice 1) MST of First-set H-2 Incompatible Grafts There are no obvious histological differences between skin allograft rejection in +/+ and W/W mice. The healing process, the i n f i l t r a t i o n of the graft by mononuclear and polymorphonuclear c e l l s , and the death and replacement of the graft appear to follow similar patterns in both groups of mice. The similarity of the MSTs for skin allografts on +/+ and W/WV mice, as determined by microscopic exami-nation, indicates that there is no marked difference in graft survival times between the two groups. The MST reported here of 9.7 days for DBA/2 skin on C57B1/6 hosts i s , however, somewhat shorter than the MST of 10.4 days reported elsewhere (Microbiological Associates, Inc., 19 72) for this donor-host combination. This discrepancy is almost certainly due to the fact that graft death w i l l be apparent microsco-pically sooner than i t w i l l be apparent macroscopically. 2) Graft Rejection Scores on the Eighth Day after Transplantation The distributions of graft rejection scores for the two groups of mice on the eighth day, however, are significantly different. This significance is not borderline, but quite marked (p = < .01). Such a 105 d i f f e r e n c e i n the d i s t r i b u t i o n of g r a f t r e j e c t i o n scores probably i n d i -cates that a d i f f e r e n c e i n the vigour of g r a f t r e j e c t i o n does i n f a c t e x i s t between the two groups of mice. In looking only f o r the end point of g r a f t s u r v i v a l as i s done i n MST determination much i n f o r -mation concerning the r e j e c t i o n process i s l o s t . The time when graf t breakdown begins, and the i n t e n s i t y of the reaction within the i n d i -v i d u a l g r a f t s during the r e j e c t i o n period may be as s i g n i f i c a n t as the end point i n comparing the r e j e c t i o n process i n experimental and c o n t r o l groups. H i s t o l o g i c a l c r i t e r i a described e a r l i e r (p. 58) d i r e c t l y r e f l e c t the vigour of the r e j e c t i o n response and the degree of damage suff e r e d by the g r a f t at any stage a f t e r t r a n s p l a n t a t i o n ; such c r i t e r i a have been used before where s e n s i t i v e comparisons of a l l o g r a f t reactions have been required (Corson et a l . , 1967). Using these c r i t e r i a , i n f o r -mation concerning the number of grafts j u s t entering breakdown and the magnitude of the reaction w i t h i n each g r a f t as w e l l as the number of g r a f t s already dead can be u t i l i z e d . An analysis of the r e j e c t i o n process which i n this way considers several aspects of the process rather than simply the end point must be a more s e n s i t i v e analysis than MST determination alone. I t i s tempting to conclude, therefore, that although there i s no d i f f e r e n c e i n MST between +/+ and W/WV mice there may i n f a c t be a d i f f e r e n c e i n the vigour and i n t e n s i t y of g r a f t v r e j e c t i o n between the two groups, with that of W/W mice being the stronger and more intense. 106 3) Rejection of Skin A l l o g r a f t s Across Weak H- Ba r r i e r s Although no differe n c e i n speed of r e j e c t i o n was apparent when H-2 incompatible graf t s were transplanted to +/+ and W/WV hosts, a d i f -ference i n the speed of r e j e c t i o n of grafts bearing weak H antigens may i n f a c t e x i s t . When ski n from male donors was transplanted to +/+ and W/WV female hosts, no su r v i v i n g g r a f t s were found among the W/WV hosts at times when grafts to +/+ hosts were almost a l l s t i l l a l i v e . These data suggest that the s u r v i v a l time of ski n transplanted v across weak H b a r r i e r s may be s i g n i f i c a n t l y shorter among W/W hosts than among +/+ hosts. 4) A Study of the C e l l u l a r I n f i l t r a t e (Collen, 1974) A study which may furth e r substantiate the more intense re-action of W/WV mice to skin a l l o g r a f t s i s that of Collen (1974). Working with mice from the same stocks used i n the experiments reported i n th i s t h e s i s , Collen placed skin a l l o g r a f t s on +/+ and W/WV hosts. At various times a f t e r transplantation, she i s o l a t e d the c e l l s i n f i l t r a t i n g the a l l o g r a f t s by enzymatically digesting the g r a f t s . She then charac-t e r i z e d the c e l l types and determined t h e i r r e l a t i v e numbers i n each g r a f t . Collen i d e n t i f i e d eight types of leukocytes w i t h i n the g r a f t s : small, large, and transformed lymphocytes; small and large hyperbaso-p h i l i c lymphoid c e l l s ; macrophages, neutrophils, and eosinophils. The r e l a t i v e numbers of these c e l l s i n gr a f t s on W/WV mice was s i g n i f i c a n t l y d i f f e r e n t from t h e i r r e l a t i v e numbers i n grafts on +/+ mice, on a l l days following transplantation. Of p a r t i c u l a r i n t e r e s t i s Collen's observation that the number of transformed lymphocytes i s s i g n i f i c a n t l y 107 higher in grafts on W/WV hosts than in grafts on +/+ hosts, on a l l days following transplantation. Transformed lymphocytes are correlated both with the i n i t i a l response of lymphoid cells to antigen and with cytotoxicity, and are thought to represent the effector arm of the allograft response. Their presence in greater numbers in allografts on W/W hosts constitutes an intriguing correlation with the more severe histological picture suggested by other experiments. Further consideration of these results and speculation as to / V the cause of the apparent differences between +/+ and W/W mice w i l l be deferred u n t i l the end of the next section of this thesis, which concerns another experiment of relevance to the present discussion. II. LYMPHOCYTE ACTIVATION STUDIES A. General Discussion Numerous studies have described the changes occurring in lymph nodes following antigenic stimulation (Scothorne, 1957; Andre et a l . , 1962; Andre-Schwartz, 1964; Coons, 1958; Parrot and de Sousa, 1966; Devlin and Ramm, 1971), and a few studies, mainly by Hall (1967) and Hall and coworkers (1963, 1967, 1971) have described changes in the c e l l population of the afferent and efferent lymph of lymph nodes draining the site of antigenic stimulation. Only a few studies exist, however, of the cellular changes in the peripheral blood following immunization. In one of the earliest of these, G i l l (1967) obtained white blood cells from normal patients and from patients with drug allergies, 108 and cultured these cells in vitro for up to five days. The numbers of cells undergoing spontaneous transformation was greater in cultures obtained from a l l e r g i c patients than in cultures from healthy subjects. G i l l suggested that this difference was-due to increased numbers of circulating, sensitized lymphocytes in the cultures from the hyper-sensitive patients. In a study of dogs bearing cardiac transplants, Tennenbaum et a l . (1970) cultured blood lymphocytes for 24 or 72 hours. Ce l l cultures from dogs bearing allografts showed a 3- to 4-fold greater percentage of spontaneously transforming lymphocytes than did c e l l cul-tures from normal dogs. Tennenbaum suggested that the increased rate of spontaneous lymphocyte transformation is a reflection of host immuno-competent cells responding to the presence of the allograft. Crowther et a l . (1969) have reported that patients with infections or under immunological stimulation also have increased numbers of "atypical mononuclear c e l l s " in the blood. These atypical cells were identified as plasma ce l l s , hyperbasophilic medium-sized lymphocytes, and large lymphoid c e l l s , and were thought to represent a population of cells derived from lymphoid tissue responding to antigenic stimulation. This conclusion is supported by Hall and Morris (1963) and Hall (1967) who described identical cells in the lymph flowing from nodes draining sites of antigenic stimulation. The numbers of atypical mononuclear cells in the blood of immu-nized patients, though greater than that of normal patients, is s t i l l low (less than 4%) (Crowther ejt a_l. , 1969). Their presence can be more readily detected by measuring the uptake of labelled DNA precursors 109 by these c e l l s . Crowther et a l . (1969) reported that about 30% of the a t y p i c a l mononuclear c e l l s i n the blood of immunologically stimu-lat e d ( patients were i n DNA synthesis, as shown by radioautographic studies of c e l l cultures l a b e l l e d with t r i t i a t e d thymidine. Hersh et a l . (1970, 1971), Dent (1970), Page et a l . (1971), and McDonald and Lee (19 74), measuring r a d i o a c t i v i t y by s c i n t i l l a t i o n counting, report a 2- to 10-fold increase i n the spontaneous uptake of t r i t i a t e d thymidine by p e r i p h e r a l blood lymphocytes of r e n a l transplant p a t i e n t s , an increase which always preceded c l i n i c a l signs of r e j e c t i o n by several days. Ono e_t a l . (1972) report s i m i l a r r e s u l t s with rats bearing heart a l l o g r a f t s . The spontaneous uptake of r a d i o a c t i v e l y l a b e l l e d DNA pre-cursors by c e l l s from the p e r i p h e r a l blood of humans or animals bearing a l l o g r a f t s appears, therefore, to r e f l e c t host immunologic r e a c t i v i t y , and to c o r r e l a t e with and precede r e j e c t i o n c r i s e s . Morphologically, the a t y p i c a l c e l l s responsible f o r the uptake of r a d i o a c t i v e l a b e l are mostly medium or large lymphoid c e l l s , many with hyperbasophilic cytoplasm (Crowther et a l . , 1969). These c e l l s are probably derived from small or medium lymphocytes that are responding to antigenic stimulation. In v i t r o experiments have shown that small lymphocytes upon stimu l a t i o n with antigen or mitogens, enter DNA syn-t h e s i s , "transform" i n t o large b a s o p h i l i c c e l l s , and eventually enter into mitosis (Ling, 1968). Lymphocytes have been shown to respond i n a s i m i l a r manner to a l l o g e n e i c c e l l s i n v i t r o , giving r i s e to large b a s o p h i l i c c e l l s that are themselves s p e c i f i c a l l y cytotoxic f o r the a l l o g e n e i c target c e l l s (Ginsburg and Sachs, 1965; Ginsburg et a l . , 110 1969; Ginsburg, 1971; Ax and Koren, 1972). The i n vivo response to antigen presumably follows a s i m i l a r pattern. The a t y p i c a l mononuclear c e l l s found i n the blood of a l l o g r a f t r e c i p i e n t s , therefore, probably represent small lymphocytes which have been ac t i v a t e d by a l l o g r a f t antigens and which are undergoing b l a s t transformation and ultimately c e l l d i v i s i o n . v B. Lymphocyte A c t i v a t i o n Studies i n +/+ and W/W Mice The uptake of a greater amount of t r i t i a t e d thymidine by p e r i -v pheral blood c e l l s of W/W mice bearing skin a l l o g r a f t s than by p e r i -pheral blood c e l l s of +/+ mice bearing a l l o g r a f t s i s good evidence that greater numbers of ac t i v a t e d lymphocytes are present i n the c i r c u l a t i o n of the mutant animals. The presence of great numbers of a c t i v a t e d lymphocytes i n these mice suggests that more c e l l s capable of responding to g r a f t antigens have been stimulated and released i n t o the c i r c u -l a t i o n . These r e s u l t s alone are strong evidence that the a l l o g r a f t reaction of W/WV mice i s somewhat more vigourous than that of +/+ mice, and as such co n s t i t u t e s u b s t a n t i a l support f o r the r e s u l t s of e a r l i e r h i s t o l o g i c a l studies which also i n d i c a t e d a more intense g r a f t reaction i n the mutant mice. C. A C o r r e l a t i o n of Observations from Lymphocyte A c t i v a t i o n Studies  and A l l o g r a f t Rejection Studies i n +/+ and W/Wv Mice The c o r r e l a t i o n between a more vigourous g r a f t r e a c t i o n i n W/W mice, as suggested by h i s t o l o g i c a l studies, a greater number of activat e d c e l l s i n the p e r i p h e r a l blood, and a greater proportion of I l l transformed c e l l s i n the c e l l u l a r i n f i l t r a t e within the g r a f t i t s e l f i s p a r t i c u l a r l y i n t r i g u i n g . The presence of greater numbers of a c t i -vated c e l l s i n the p e r i p h e r a l blood of W/WV mice bearing s k i n a l l o g r a f t s suggests that g r a f t antigens have succeeded i n a c t i v a t i n g greater numbers of lymphoid c e l l s i n the mutant hosts, c e l l s which are then released from the lymph nodes in t o the c i r c u l a t i o n . I f greater numbers of a c t i -vated c e l l s are present i n the p e r i p h e r a l blood, then greater numbers of s e n s i t i z e d c e l l s w i l l also be present among the c e l l s i n f i l t r a t i n g skin a l l o g r a f t s , even i f the l o c a l i z a t i o n of c e l l s within those gra f t s i s random rather than s p e c i f i c . Once i n the g r a f t , these s e n s i t i z e d c e l l s may i n t e r a c t with the c e l l s of the g r a f t and exert a d i r e c t cyto-tox i c e f f e c t ; they probably also i n t e r a c t with g r a f t antigens and release lymphokines. I f greater numbers of s e n s i t i z e d c e l l s are present i n the g r a f t then greater q u a n t i t i e s of lymphokines may be produced, and the reactions mediated by these substances w i l l be correspondingly more intense. Thus, greater numbers of macrophages and eosinophils may accumulate i n the g r a f t i n response to MIF and ECF, greater numbers of unsensitized lymphocytes may be transformed by LTF and TF, and greater numbers of target c e l l s may be damaged by lymphotoxin. Whether or not the c e l l u l a r i n f i l t r a t e i s indeed greater, i n terms of absolute numbers of c e l l s , i n grafts on W/W hosts, has not been determined. That the proportion of transformed lymphocytes i n the g r a f t s , however, i s greater, and the h i s t o l o g i c a l p i c t u r e somewhat more severe, and that these para-meters are c o r r e l a t e d with increased numbers of a c t i v a t e d c e l l s i n the p e r i p h e r a l blood, suggests that the number of c e l l s able to respond to g r a f t antigens may be a key differ e n c e between +/+ and W/Wv mice, and may be a s i g n i f i c a n t parameter i n determining the i n t e n s i t y of the gra f t reaction. W/WV mice appear to r e j e c t weakly antigenic s k i n g r a f t s i n less time than do +/+ mice, although the r e j e c t i o n of strongly a n t i -genic g r a f t s does not appear to be fa s t e r than normal. This d i f f e r e n -t i a l response to strong and weak antigens i s perhaps also most r e a d i l y v explained i n terms of the numbers of c e l l s i n +/+ and W/W mice that are reactive to strong and weak gr a f t antigens. McGregor and Gowans (1964) and Roser and Ford (19 72) have r e -ported that the s u r v i v a l time of sk i n a l l o g r a f t s exchanged across major h i s t o c o m p a t i b i l i t y b a r r i e r s i s only marginally reduced i n lymphocyto-penic mice; lymphocytopenia has a more marked e f f e c t , however, when only minor h i s t o c o m p a t i b i l i t y differences e x i s t between donor and host. The reason f o r the d i f f e r e n t i a l e f f e c t of lymphocyte depletion on strong and weak a l l o g r a f t reactions i s not known. Roser and Ford (1972), however, c i t e evidence that the number of c e l l s r e a c t i v e with strong antigens i s much greater than the number of c e l l s r e a c t i v e with weak antigens, and speculate that even i n lymphocytopenic animals the concen-t r a t i o n of c e l l s able to respond to strong antigens may be greater than the concentration of c e l l s r eactive to weak antigens i n untreated animals. They go on to suggest that above a c r i t i c a l concentration of antigen-sensitive c e l l s an increase i n the concentration of c e l l s has l i t t l e e f f e c t on skin g r a f t s u r v i v a l time (which may be dependent on the mini-mum time required to generate e f f e c t o r c e l l s ) , but below th i s concentration the s u r v i v a l time depends on the concen-t r a t i o n of antigen-sensitive c e l l s . 113 v Results of a l l o g r a f t r e j e c t i o n i n W/W mice suggest a s i m i l a r hypothesis. Although the r e j e c t i o n of H-2 incompatible graf t s i n W/WV mice i s associated with greater numbers of activat e d c e l l s i n the p e r i -pheral blood, with a greater proportion of transformed c e l l s within the grafts themselves, and with a generally more severe h i s t o l o g i c a l p i c t u r e , the MST of these grafts i s not reduced, po s s i b l y because, as Roser and Ford suggest, beyond a c e r t a i n c r i t i c a l concentration of reactive c e l l s , an increase i n the numbers of reac t i v e c e l l s simply has no e f f e c t on g r a f t s u r v i v a l times. I t may not be poss i b l e to reduce the s u r v i v a l time of f i r s t - s e t a l l o g r a f t s below about eight or nine days, a l i m i t probably dependent on the minimum time required f o r heal i n g , antigen recognition, s e n s i t i z a t i o n , and the production of e f f e c t o r c e l l s . A l l o g r a f t s bearing weak anti'gens, however, survive two to three times longer than do H-2 incompatible g r a f t s , perhaps because they normally stimulate a much smaller number of re a c t i v e c e l l s , a number below the c r i t i c a l number required to cause g r a f t r e j e c t i o n i n minimum time. Differences i n the concentration of rea c t i v e c e l l s between groups of animals bearing weakly antigenic g r a f t s may, therefore, have s i g n i -f i c a n t e f f e c t s on g r a f t s u r v i v a l times: an increase i n the number of r e a c t i v e c e l l s might s i g n i f i c a n t l y decrease g r a f t s u r v i v a l time. In summary: W/WV mice may have greater numbers of c e l l s able to respond to g r a f t antigens than do +/+ mice, but an increase i n the numbers of reac t i v e c e l l s has no e f f e c t on s u r v i v a l times of strongly antigenic g r a f t s , which normally a c t i v e many more than the c r i t i c a l number of antigen-sensitive c e l l s . In the case of weakly antigenic 114 g r a f t s , however, which normally a c t i v a t e fewer antigen-sensitive c e l l s and are rejected slowly, the presence of greater numbers of responsive v c e l l s i n W/W mice s i g n i f i c a n t l y shortens g r a f t s u r v i v a l time. I I I . HUMORAL IMMUNE RESPONSES A. General Discussion Lymphocytes have been implicated i n the production of antibody at l e a s t since 1935 when McMaster and Hudack demonstrated that the formation of b a c t e r i a l agglutinins took place i n lymph nodes. Many workers (e.g. Dougherty et a l . , 1944; Harris eit a_l. , 1945) belie v e d that the antibody-forming c e l l (AFC) was the small lymphocyte, although the general consensus held that plasma c e l l s were the antibody producers (Bjorneboe and Gormsen, 1943; Fagraeus, 1948; Yoffey, 1964). In 1955, Leduc et_ a_l. , using the now c l a s s i c fluorescent antibody technique of Coons et a l . (1955), succeeded i n demonstrating beyond doubt that antibodies were produced by plasma c e l l s . Later studies, however, made i t c l e a r that lymphocytes, too, were e s s e n t i a l f o r antibody syn-t h e s i s : Gowans et a l . (1962) showed that rats depleted of lymphocytes by chronic drainage of the thoracic duct gave a severely depressed response to antigen, and that t h i s response was restored by i n j e c t i o n s of small lymphocytes. Gowans (1962) also demonstrated that lymphocytes could change into d i v i d i n g c e l l s with new morphological c h a r a c t e r i s t i c s , c e l l s resembling those seen i n regional lymph nodes during antibody formation, and he suggested that the small lymphocyte might be the 115 precursor of the plasma c e l l . Other work (Harris et a l , , 1966; McGregor et. a l . , 1967; Birbeck, 1967) has provided more d e f i n i t i v e evidence of the transformation of lymphocytes in t o plasma c e l l s . Weiss (19 72) now considers i t " l i k e l y , " and R o i t t et d . (1969) consider i t " c l e a r " that c e r t a i n small lymphocytes can respond to antigen by enlarging,pro-l i f e r a t i n g , and producing a population of antibody-forming plasma c e l l s . Recent studies using s p e c i a l techniques f or the l o c a l i z a t i o n of antibody by el e c t r o n microscopy have demonstrated antibodies i n both lymphocytes and plasma c e l l s , and have presented perhaps the cle a r e s t p i c t u r e of the c e l l s involved i n antibody synthesis. Antibody appears to be formed i n t y p i c a l plasma c e l l s , i n some large lymphocytes, and i n c e r t a i n c e l l s ("lymphoplasmacytes") which have some morphological c h a r a c t e r i s t i c s of both plasma c e l l s and small lymphocytes (Avrameas and B o u t e i l l e , 1968; Leduc et a l . , 1968; Avrameas and Leduc, 1970). Lymphocytes, although morphologically homogeneous, are known to comprise at l e a s t two d i s t i n c t subpopulations, a thymus-dependent population and a thymus-independent population; these are designated r e s p e c t i v e l y T - c e l l s and B - c e l l s (see R o i t t et a l . , 1969; Raff, 1970) and carry c h a r a c t e r i s t i c surface antigen markers. Those c e l l s respon-s i b l e f o r the production of humoral antibodies belong to the B - c e l l population ( f o r review see Strober, 19 75). Experiments summarized by Weiss (1972) have demonstrated that some sort of cooperation between lymphocytes and macrophages or between d i f f e r e n t populations of lymphocytes i s necessary f or the production of antibodies to c e r t a i n antigens. Antibody-forming B - c e l l s seem to 116 require some sort of stimulation from macrophages or from antigen-sensitive T-cells. Feldmann (19 73) and Byrd et a l . (19 74) have noted that those antigens which are T-cell dependent are generally monomeric antigens, and have suggested that the role of T-cells and macrophages in the antibody response is to bind these antigen molecules and present them to B-cells in a locally concentrated form. There is evidence that T-cells produce a soluble immunoglobulin-like factor which binds antigen and is cytophilic for macrophages, rendering them capable of s p e c i f i -cally immunizing B-cells (Feldmann, 1973). This model, however, is speculative, and the nature of c e l l cooperation in the antibody response s t i l l controversial and far from clear. Other cellular events in the process of antibody synthesis are also s t i l l far from being understood. For some time, however, i t has been taken for granted that immunoglobulin receptors on B lympho-cytes are involved in antigen recognition by these c e l l s , and in B-c e l l activation (Greaves, 19 70; Nossal et a l . , 19 72; Hammerling and McDevitt, 1974a, 1974b; Kreth and Herzenberg, 1974). Further, i t is generally thought that in the i n i t i a l or primary response to an antigen such as SRBC, interaction with antigen causes a small proportion of specifically reactive cells in lymphoid organs to undergo clonal expan-sion, increasing in number perhaps 1,000 times and giving rise to cells which secrete specific antibodies, most of which are IgM (Shearer et a l . , 1968; Wilson and Nowell, 1971). Upon second exposure to antigen, the greater proportion of antigen-reactive cells which now exists is be-lieved to result in the prompt and prolonged production of even higher 117 t i t e r s of antibody, although the antibody now produced i s l a r g e l y IgG (Wilson and Nowell, 19 71). I n d i v i d u a l AFC are characterized by the production of d i f f e r e n t types of antibody, i n d i v i d u a l c e l l s s e c r e t i n g antibody of only one type. Antibodies capable of l y s i n g SRBC d i r e c t l y are probably of the IgM c l a s s , and those capable of a g g l u t i n a t i o n or i n d i r e c t l y s i s are probably of the IgG class (Wortis e_t a l . , 1966). Whether c e l l s producing d i f f e r e n t types of antibody a r i s e from common precursors ( S t e r z l and Nordin, 19 71) or from unipotent precursors (Shearer et a l . , 1968, 1969a, 1969b) i s uncertain. I n d i v i d u a l AFC can be detected by the plaque formation technique, whereby spleen c e l l s are mixed with SRBC and plated e i t h e r i n agar or i n suspension between glass s l i d e s . Target c e l l s surrounding i n d i v i d u a l AFC are lysed w i t h i n an hour or so by antibody secreted by active c e l l s . Cunningham (1968b) reports- a v a r i e t y of morphological types among mouse AFC detected by the plaque technique: c l a s s i c a l mature plasma c e l l s account for a small number of plaques, but most AFC are " b a s o p h i l i c mononuclears with r e l a t i v e l y l i t t l e cytoplasm." Cunningham discerns no consistent morphological d i f f e r e n c e between c e l l s r e l e a s i n g IgM and those r e l e a s i n g IgG. The absolute number of AFC detected by the plaque technique, and the time of peak response varies with the dose of antigen and the route of administration (Cunningham, 1968b; Wortis e_t a l . , 1966). I n t r a p e r i t o n e a l administration r e s u l t s i n a peak response of d i r e c t plaques by the fourth day (Wortis ejt a l . , 1966) . 118 B. Humoral Immune Responses in +/+ and W/WV Mice The number of spleen cells actively secreting SRBC hemolysins in W/WV mice during the primary response to antigen is about 1/4 that in +/+ mice, as determined by plaque assay. The concentration of SRBC agglutinins produced by W/WV mice, however, i s essentially identical to that produced by +/+ mice. W/WV mice might appear, therefore, to be defective in hemolysin (IgM) production but not in hemagglutinin (IgG) production. This di f f e r e n t i a l defect, however, may not reflect differences in immunoglobulin concentration so much as differences in the sensitivity of the assays used. A small difference in agglutinin concentration would not be detected by the s e r i a l dilution technique which, at best, is subject to a 2-fold error. In the secondary response to antigen a small but significant difference between antibody titers of +/+ and W/WV mice indicates that hemagglutinin production in the mutant mice is indeed defective. A defect in PFC numbers in W mutant mice has been reported before: Shearer and Cudkowicz (196 7) found that WV/WV mice produced only 1/3 to 1/2 the number of PFC as did their WV/+ littermates; this ratio agrees well with the results reported in this thesis. Another study, however, finds no difference between +/+ and W/WV mice in the PFC response to SRBC (Mekori and P h i l l i p s , 1969); indeed, in this study a slightly higher PFC response was found among W mutants than among coisogenic normal mice. An explanation for the lack of agreement among the results from different laboratories is not readily apparent. Mekori and 119 P h i l l i p s used W/WV mice while Shearer and Cudkowicz used mice homozy-gous f o r WV; Mekori and P h i l l i p s suggest, therefore, that the d i f f e r e n t combination of a l l e l e s may account for the d i f f e r e n t r e s u l t s . This v hypothesis, however, does not appear to hold: W/W mice, as reported i n this t h e s i s , as w e l l as by McMaster (1973), can also produce a defec-t i v e PFC response. There i s , therefore, no obvious d i f f e r e n c e between the experiments conducted i n the d i f f e r e n t l a b o r a t o r i e s which might account for the d i s p a r i t y i n r e s u l t s . A number of mechanisms can be suggested to explain the defec-t i v e response of W/WV mice to SRBC antigens: 1) reduced numbers of antigen-reactive B - c e l l s ; 2) a defect i n the a c t i v i t y of the helper c e l l s , e i t h e r T - c e l l s or macrophages, that are necessary f o r a normal response to SRBC; 3) a defect i n the i n t e r a c t i o n between the B - c e l l and the antigen, such that a c t i v a t i o n of B - c e l l s i s i n h i b i t e d ; 4) a defect i n the a c t i v a t i o n mechanism i t s e l f , such that fewer B - c e l l become activated; 5) a defect i n p r o l i f e r a t i o n , such that fewer AFC are produced; 6) defective immunoglobulin production. In view of what appears to be at l e a s t normal T - c e l l function i n the a l l o g r a f t response, a helper c e l l defect seems perhaps the l e a s t l i k e l y of the above hypotheses. Shearer and Cudkowicz (1967), however, have reported that spleens of homozygous WV mice contain about h a l f the number of antigen-reactive c e l l s (ARC) (as' determined by focus assay) as do WV/+ mice. This report suggests that those ARC which are a c t i v a t e d v . v i n W /W mice p r o l i f e r a t e normally and produce normal q u a n t i t i e s of 120 immunoglobulins. Hypothesis 1) above appears, therefore, to be sup-ported, and hypotheses 5) and 6) to be u n l i k e l y . A deficiency i n numbers of ARC may be due to the absence of these c e l l s or to the i n a b i l i t y of the c e l l s present to react to st i m u l a t i o n , as suggested by hypotheses 3) and 4). Present knowledge does not allow a choice between these p o s s i b i l i t i e s , but hypothesis 3) w i l l be discussed i n more d e t a i l l a t e r . IV. MITOGEN STIMULATION STUDIES A. General Discussion Small lymphocytes can be induced by treatment with various mitogenic agents to undergo c e r t a i n metabolic and.morphologic changes, ultimately r e s u l t i n g i n the formation of b l a s t - l i k e c e l l s . This reac-t i o n was f i r s t reported by Nowell (1960), who found that the plant l e c t i n phytohaemagglutinin (PHA) induced transformation of p e r i p h e r a l blood lymphocytes. Other plant l e c t i n s such as concanavalin-A (Con-A) and pokeweed mitogen (PWM), and agents such as b a c t e r i a l endotoxins (LPS), have since been reported to have s i m i l a r e f f e c t s (Fames et a l . , 1964; Powell and Leon, 1970). The induction of b l a s t transformation by n o n s p e c i f i c mitogens i s not an immunological response, but i s of p a r t i c u l a r i n t e r e s t because i t appears to mimic the response of lymphocytes to antigen. Lympho-cytes from unsensitized i n d i v i d u a l s , f o r example, w i l l respond to the presence of al l o g e n e i c lymphocytes iti v i t r o by undergoing b l a s t trans-formation (Bain e_t al. , 1963, 1964). Lymphocytes w i l l also respond 121 in vitro to other types of cells bearing foreign transplantation anti-gens, such as embryonic c e l l monolayers (Ax and Koren, 1972; Ginsburg et a l , , 1969; Altman ejt _al. , 1973), and cells of lymphoid lines (Hardy et a l . , 1969). In addition, lymphocytes from sensitized individuals w i l l undergo marked•transformation and proliferation i f exposed i n vitro to the sensitizing antigen (Mills, 1966). The blast cells arising from unsensitized lymphocytes responding to allogeneic c e l l s , and from sensitized lymphocytes cultured with specific antigens are, superfi-c i a l l y at least, similar to those blast cells arising from lymphocytes stimulated non-specifically with plant lectins. In addition, these blast cells closely resemble those transformed or activated lymphocytes which appear in the lymph and peripheral blood of animals bearing a l l o -grafts (p.109). Nonspecific mitogenic stimulation, therefore, i s gener-al l y considered not only a model useful in analyzing the events i n -volved in lymphocyte activation, but also a means for monitoring the immunological competence of lymphocytes from test subjects (Janossy and Greaves, 19 71). The morphological and ultrastructural changes occurring after mitogenic stimulation have been described in detail (Ling, 1968). Briefly, these involve a marked increase in the nuclear-cytoplasmic ratio, the appearance of a highly developed Golgi apparatus and more abundant mitochondria, and increased cytoplasmic basophilia related to increased numbers of ribosomes. RNA synthesis, protein synthesis, and eventually DNA synthesis occur, and after two or three days of treatment mitoses occur. 122 Bla s t c e l l s produced upon lymphocyte transformation appear to be cytotoxic for target c e l l s . The b l a s t c e l l s formed from lymphocytes cultured on monolayers of fo r e i g n c e l l s (Ax and Koren, 1972; Ginsburg, 1971; Ginsburg and Sachs, 1965; Ginsburg et a l . , 1969), with s u b c e l l u l a r h i s t o c o m p a t i b i l i t y antigens (Koldovsky e_t a l . , 1969; Manson and Simmons, 1969), or i n some mixed lymphocyte cultures (Andersson and Hayry, 1973; Hodes and Svedmyr, 19 70) are reported to be s p e c i f i c a l l y cytotoxic f o r c e l l s of the s e n s i t i z i n g genotype. Activated c e l l s a r i s i n g from lympho-cytes stimulated with plant l e c t i n s are also reported to be c y t o t o x i c for target c e l l s (Holm and Perlmann, 1965; S t e j s k a l e t a l , , 1973), but Perlmann and Holm (1969) and others (e.g. Asherson et a l . , 1973; Warnatz et a l . , 1974) emphasize that t h i s c y t o t o x i c i t y i s n o n s p e c i f i c . There i s some question as to whether the b l a s t c e l l s produced upon mitogen stim u l a t i o n and the cyto t o x i c e f f e c t o r c e l l s seen i n these cultures a r i s e from the same population of lymphocytes ( S t i t e s et a l . , 1972; D a g u i l l a r d , 1972). Although degree of stim u l a t i o n and degree of c y t o t o x i c i t y usually c o r r e l a t e w e l l , the transformed c e l l s may not themselves be the e f f e c t o r c e l l s ( S t e j s k a l e_t ail. , 1973). Indeed, recent reports i n d i c a t e that the generation of c y t o t o x i c e f f e c t o r lympho-cytes need not c o r r e l a t e at a l l with mitogen-induced transformation or with mixed lymphocyte r e a c t i v i t y , and i s i n f a c t independent of the p r o l i f e r a t i v e response (Rocklin, 1972; Mawas e_t al. , 1975; Corley e_t. a l . , 19 75). The hypothesis that antigens a c t i v a t e lymphocytes v i a receptors on the lymphocyte surface i s now generally accepted (see Diener and 123 Langman, 1975, for review). Normal mouse lymphocytes appear to carry s p e c i f i c surface receptors for antigens (Hammerling and McDevitt, 19 74), i n c l u d i n g h i s t o c o m p a t i b i l i t y antigens (Altman et a l , , 1973). Consider-able evidence i n d i c a t e s that these receptors, at l e a s t i n B - c e l l s , are immunoglobulins (Melchers and Andersson, 1973; Marchalonis et a l . , 1972; Marchalonis and Cone, 1973; Nossal e_t a l . , 1972; Pernis et a l . , 19 74; V i t e t t a et a l . , 19 71); the nature of the T - c e l l receptor i s s t i l l c o n t r o v e r s i a l . Nonspecific mitogens such as the plant l e c t i n s also appear to a c t i v a t e lymphocytes v i a surface receptors. PHA has been reported to l o c a l i z e within c e l l s (Stanley et a l , , 1969; Rubin and Schultz, 1973), but the i n i t i a l event at l e a s t involves a membrane receptor. Polgar e_t al_. (1969) report a d i r e c t r e l a t i o n s h i p between the binding of PHA to c e l l s and the metabolic a l t e r a t i o n s which follow, and A l l a n et a l , (1971) report that the mitogenic a c t i v i t y of PHA can be e f f e c -t i v e l y adsorbed by plasma membranes of lymphocytes and thymus c e l l s . The nature of the PHA receptor has not been determined, but i s c o n s i -dered most l i k e l y to be a glycoprotein (Daguillard, 1972). Although lymphocytes carry s p e c i f i c immunoglobulin receptors for the antigenic determinants of LPS, evidence i n d i c a t e s that these are u n l i k e l y to be the receptors responsible f o r n o n s p e c i f i c a c t i v a t i o n , LPS receptors, however, have not been characterized (Andersson et a l . , 1972a). Con-A, on the other hand, appears to bind s e l e c t i v e l y to methyl alpha-D-mannoside i n the lymphocyte surface (Powell and Leon, 19 70; Betel and van den Berg, 1972). The a d d i t i o n of these sugars to the 124 culture system reduces the binding of Con-A to the c e l l , but does not affect the binding of PHA (Powell and Leon, 19 70). The i n i t i a l event in lymphocyte activation by mitogen, therefore, appears to involve surface receptors, and these receptors are probably mitogen-specific. The mechanism by which non-specific mitogens activate, lymphoid cells i s not known. Although PHA has been reported to enter the nucleus and bind to heterochromatin (Rubin and Schultz, 1973; Stanley et a l . , 1969), most reports indicate that prolonged interaction of mitogen with the c e l l surface receptor is sufficient to induce transformation (Betel and van den Berg, 1972), Activation of chromatin, then, may occur through one or several secondary messengers (Skoog et a l . , 19 73) possibly involving cyclic-AMP (Krishnaraj and Talwar, 19 73). PHA and Con-A are reported to cause as much as a 3-fold increase in cAMP levels i n lymphocytes within one minute of incubation (Parker, 1974). Indeed, Watts (19 71) has proposed that the activation of cells by antigen may be analogous to the action of hormones on c e l l s , involving cAMP as a secondary messenger. Although there is thus reason to suspect that cyclic nucleotides are involved in lymphocyte activation, their role i s not yet firmly established, and the mechanism of mitogen activation remains unclear. Different mitogens appear to activate different populations of small lymphocytes. In mice, for example, PHA and Con-A appear to be selectively active on T-cells, and LPS selectively active on B-cells (Andersson et a l . , 1972a; Peavy et a l . , 1974; Janossy and Greaves, 1971). The basis for this selective activation, however, is not known. 125 The presence or absence of appropriate receptors may be relevant i n some instances, but i n the case of Con-A, f o r example, B- and T - c e l l s appear to carry equal numbers of receptors and to bind Con-A to the same extent (Moller et a l . , 19 73). B - c e l l a c t i v a t i o n by Con-A appears, therefore, to require more than binding of Con-A to the surface. Models have been proposed to explain s e l e c t i v e a c t i v a t i o n i n terms of c e l l cooperation or requirements for soluble substances a f t e r binding has taken place, but these are s t i l l h ighly speculative and c o n t r o v e r s i a l (Andersson, et a l . , 1972a, 19 72b). B. Mitogen Stimulation Studies i n +/+ and W/WV Mice The response of spleen c e l l s from W/W mice to the mitogen LPS i s c l e a r l y d e f i c i e n t : both raw count means and mean sti m u l a t i o n indices of W/WV cultures are always lower than those of +/+ c u l t u r e s , regardless of the concentration of mitogen used or the length of time i n c u l t u r e (the d e f i c i e n c y , however, i s most marked at longer culture periods). A number of hypotheses may be proposed to explain the d e f i -v c i e n t response of W/W spleen c e l l s to LPS: 1. a smaller proportion of B - c e l l s and therefore a smaller proportion of LPS-responsive c e l l s i n W/Wv c u l t u r e s ; 2. a deficiency of LPS receptors on W/WV c e l l s with a conse-quent deficiency i n the binding of LPS to W/Wv c e l l s ; 3. i n a b i l i t y of bound LPS to stimulate transformation i n W/WV c e l l s . Present knowledge does not allow a choice between these p o s s i b i l i t i e s . v The response of spleen c e l l s from W/W mice to the plant l e c t i n Concanavalin-A appears to be marginally stronger than the response 126 of +/+ spleen c e l l s to t h i s mitogen. Stimulation indices for cultures of W/WV c e l l s are not at a l l d i f f e r e n t from those f o r cultures of +/+ c e l l s , but, i f the raw counts for the two samples are compared, counts i n W/WV cultures appear higher at 72 hours than do counts i n +/+ c u l -tures, and at 96 hours, when the optimal response to Con-A i s reached, t h i s d i f f e r e n c e i s s t a t i s t i c a l l y s i g n i f i c a n t . Several hypotheses may v be proposed to explain the enhanced response of W/W c e l l s to Con-A: 1. a greater proportion of T - c e l l s and therefore a greater proportion of Con-A responsive c e l l s i n W/Wv cu l t u r e s ; 2. some a l t e r a t i o n i n the surface character of W/WV c e l l s which r e s u l t s i n optimal binding of the l e c t i n and thereby optimal s t i m u l a t i o n ; 3. increased s e n s i t i v i t y of W/WV Con-A binding c e l l s to transformation. Present knowledge does not allow a choice between these p o s s i b i l i t i e s . The c o r r e l a t i o n between depressed antibody production and depressed responses to LPS i n W/WV mice suggests a general d e f i c i e n c y i n B - c e l l responses. Likewise, the c o r r e l a t i o n between enhanced g r a f t r e j e c t i o n and p o s s i b l y enhanced responses to Con-A i n these mice suggests a general enhancement of T - c e l l responses. Further studies using other T - c e l l and B - c e l l mitogens and other T - c e l l and B - c e l l antigens would be required to determine whether or not this g e n e r a l i z a t i o n does, i n f a c t , hold. The p o s s i b i l i t y of a l t e r e d lymphocyte r e a c t i v i t y , however, suggests an i n t r i g u i n g hypothesis which w i l l be discussed i n more d e t a i l l a t e r . 127 V. OTHER STUDIES A. Macrophage Migration Studies v The observation that primordial germ c e l l s i n W/W embryos v are severely retarded i n migration, and that lymphocytes from W/W mice also migrate more slowly than normal, suggested that other migra-tory c e l l s i n these mice might be a f f e c t e d i n a s i m i l a r manner. The migration of macrophages from c a p i l l a r y tubes i n c u l t u r e , and the i n h i b i t i o n of t h i s migration by the presence of s e n s i t i z e d lymphocytes and antigen, has often been used as an i n v i t r o c o r r e l a t e of c e l l u l a r h y p e r s e n s i t i v i t y (see Friedman et a l . , 1969, f o r references). The c a p i l l a r y tube culture system seemed to o f f e r a simple method of determining whether or not the W gene exerts any gross e f f e c t on macro-phage migration. Experiments demonstrated no d i f f e r e n c e i n migration between p e r i t o n e a l exudate c e l l s from +/+ and from W/WV mice. The c a p i l l a r y tube culture method, however, proved to be highly v a r i a b l e and not, therefore, p a r t i c u l a r l y s e n s i t i v e ; small but s i g n i f i c a n t d i f -ferences i n migration between experimental and c o n t r o l groups would, therefore, not be detected by t h i s procedure. These experiments lead only to the conclusion that the W gene exerts no gross e f f e c t on macro-phage migration. B. Lymphocyte Adhesion Studies. Lymphocytes are not adhesive i n the way that macrophages, f i b r o -b l a s t s , and other c e l l s which grow i n cu l t u r e are. This lack of adhe-siveness allows them to be e a s i l y separated from such c e l l s as macrophages 128 simply by allowing a mixed c e l l population to s e t t l e on glass for h a l f an hour; macrophages w i l l adhere f i r m l y to the glass and the lympho-cytes can be washed away. Lymphocytes, are, however, somewhat " s t i c k y " i n that a c e r t a i n percentage of them w i l l adhere gently to glass and w i l l remain attached against t h e force of gravity unless f o r c e f u l l y washed away. The a b i l i t y of c e l l s to adhere to surfaces i s one very simple method of i n v e s t i g a t i n g c e r t a i n c e l l surface p r o p e r t i e s . The treatment of the c e l l s themselves, or of the surfaces on which they w i l l be placed, with various agents, determines the proportion of adherent c e l l s and helps to i n d i c a t e what factors influence c e l l adhesion. Such studies, though l i m i t e d i n what they can demonstrate, do provide a c e r t a i n amount of information about the surface properties of c e l l s . C e l l s must adhere to surfaces i n order to migrate, and i t seems a reasonable assumption that i f adhesion i s e i t h e r too great or too weak, migration w i l l be hindered. The speculation was advanced, there-fore, that the retarded migration of lymphocytes and c e r t a i n other c e l l s i n W/WV mice may be associated with some a l t e r a t i o n i n the c e l l surface, such that adhesion, and thereby migration, i s a f f e c t e d . The weak adhesion of lymphocytes to glass was used as a simple method of determining the degree of adhesiveness of +/+ and W/WV spleen c e l l populations. Tests showed, however, no differences between the pro-portions of adhesive c e l l s i n mutant and non-mutant mice. 129 VI. HAEMATOLOGY No differences are apparent between +/+ mice and mice carrying the W mutation with regard to t o t a l and d i f f e r e n t i a l white blood c e l l counts, e i t h e r before or a f t e r g r a f t i n g . Adult W/WV mice, as reported by Chervenick and Boggs (1969), are c l e a r l y capable of maintaining normal numbers of c i r c u l a t i n g granulocytes and lymphocytes, and appear to respond to transplantation procedures i n the same manner as do normal mice. The following discussion w i l l therefore make no reference to the genotypes of the mice involved. Mice bearing s k i n autografts or a l l o g r a f t s demonstrate markedly depressed white c e l l counts. S t r i k i n g v a r i a b i l i t y i n white c e l l count i n mice i s not unusual, and the changes associated with s k i n trans-p l a n t a t i o n probably r e f l e c t , at l e a s t i n part, the e f f e c t s of anaes-thes i a and the general trauma r e s u l t i n g from handling and the trans-p l a n t a t i o n process i t s e l f . In mice bearing s k i n a l l o g r a f t s , however, d i f f e r e n t i a l counts are also changed, demonstrating a marked decline i n the r a t i o of c i r c u l a t i n g lymphocytes. The lymphocytopenia asso-ci a t e d with a l l o g r a f t r e j e c t i o n has never been f u l l y explained, but may be a r e f l e c t i o n of the movement of large numbers of lymphocytes in t o the a l l o g r a f t . The observation that lymphocytopenia i s greatest a f t e r day s i x or seven, when the c e l l u l a r i n f i l t r a t e i n the a l l o g r a f t i s reaching a peak, i s consistent with t h i s hypothesis. 130 VII. SPECULATION: THE PRIMARY SITE OF ACTION OF THE W GENE Let me now summarize the r e s u l t s of t h i s study of immune v responses i n W/W mice, and speculate on an explanation f o r these r e s u l t s and on a hypothesis for the primary s i t e of action of the W gene. With regard to immune responses i n W/W mice, i t appears that: 1) weakly antigenic s k i n g r a f t s are rejected more quickly by W/WV mice than by +/+ mice. Grafts bearing strong antigens are not rejected more quickly but e l i c i t a somewhat more intense inflammatory response that i s detectable h i s t o l o g i c a l l y during the acute r e j e c t i o n period; 2) g r a f t r e j e c t i o n i n W/WV mice i s associated with greater numbers of a c t i v a t e d c e l l s i n the p e r i p h e r a l blood, and, according to Collen (1974), with a l a r g e r proportion of transformed lymphocytes wi t h i n the g r a f t i t s e l f ; 3) spleen c e l l s from W/WV mice produce fewer plaque-forming c e l l s i n response to SRBC than do spleen c e l l s from +/+ mice; 4) spleen c e l l s from W/WV mice e x h i b i t depressed B - c e l l responses and, p o s s i b l y , marginally enhanced T - c e l l responses when tested with B - c e l l and T - c e l l mitogens. Observations 1 and 2 are most r e a d i l y explained by the hypo-v thesis that W/W mice contain greater numbers of c e l l s r e a c t i v e with g r a f t antigens than do +/+ mice. Observation 3 suggests that W/WV mice contain fewer c e l l s able to respond to SRBC antigens than do +/+ mice. Since the g r a f t reaction i s p r i m a r i l y a T - c e l l response, and 131 antibody production a B - c e l l response, these observations lead to the speculation that T - c e l l responses generally may be somewhat enhanced / v i n W/W mice and B - c e l l responses depressed. This speculation gaxns some support from observation 4 , which indicates that the response to B - c e l l mitogens i n W/WV mice i s depressed and the response to T - c e l l mitogens perhaps marginally enhanced. Enhanced T - c e l l responses and depressed B - c e l l responses may be explained by po s t u l a t i n g the existence of an a l t e r e d T - c e l l / B - c e l l r a t i o i n W/WV mice: perhaps the proportion of T - c e l l s i s higher and the proportion of B - c e l l s lower i n these mice than i n normal mice. An attempt to determine the T - c e l l / B - c e l l r a t i o i n the spleens of +/+ and W/WV mice by l a b e l l i n g B - c e l l s with fluorescein-conjugated a n t i -immunoglobulin, however, has not yet y i e l d e d r e s u l t s . An a l t e r n a t i v e explanation to an a l t e r e d T - c e l l / B - c e l l r a t i o i s that normal numbers of T- and B - c e l l s i n W/WV mice are simply a l t e r e d i n t h e i r r e a c t i v i t y with antigens, T - c e l l r e a c t i v i t y being enhanced and B - c e l l r e a c t i v i t y being depressed. Speculation on t h i s p o s s i b i l i t y leads to a hypothesis which may be able to explain not only the anoma-lous immune responses of W mice, but a l l the other known e f f e c t s of the W gene as w e l l . The response of lymphocytes to antigens and to mitogens i s bel i e v e d to be i n i t i a t e d by the i n t e r a c t i o n of the sti m u l a t i n g molecule with a receptor on the lymphocyte surface. Erythropoiesis and leuko-p o i e s i s are regulated by hormones which are also known to exert t h e i r e f f e c t s v i a c e l l surface receptors. Primordial germ c e l l s -and 132 prospective pigment c e l l s may also require some external s t i m u l a t i o n , perhaps encountered as they migrate from t h e i r s i t e of o r i g i n to t h e i r s i t e i n the p e r i p h e r a l t i s s u e , i n order to complete t h e i r d i f f e r e n -t i a t i o n . Although very l i t t l e i s known about what properties of the c e l l surface influence migration, i t i s c l e a r that migration, too, i s a phenomenon i n which i n t e r a c t i o n between the c e l l surface and the environment i s c r i t i c a l . I t seems reasonable to propose, therefore, that the observed e f f e c t s of the W gene may be due to an a l t e r a t i o n i n the surface c h a r a c t e r i s t i c s of the a f f e c t e d c e l l s , such that the a b i l i t y of these c e l l s to respond to external stimulation i s a l t e r e d . The p o s s i b i l i t y that the W locus controls some c h a r a c t e r i s t i c of the c e l l surface has been suggested before (Keighley et a l , , 1966). W/WV marrow i s normally r e l a t i v e l y unresponsive even to 150x the con-centration of e r y t h r o p o i e t i n that induces massive erythropoiesis i n +/+ mice. Keighley e_t al. (1966) , however, report that prolonged hypoxia renders W/WV e r y t h r o i d stem c e l l s almost f u l l y responsive to erythro-p o i e t i n . They suggest that low oxygen tension i t s e l f may have some v d i r e c t e f f e c t on the surface of W/W e r y t h r o i d precursors, perhaps exposing or inducing the formation of e r y t h r o p o i e t i n receptors. Although a number of other hypotheses can be proposed to explain the e f f e c t of hypoxia on e r y t h r o p o i e s i s , only the " c e l l surface hypothesis," which implies that the primary a c t i o n of the W gene has to do with c e l l s t r u c -ture, i s fundamental enough and broad enough to account f o r the other v observed anomalies of W/W mice as w e l l . Changes i n c e l l surface c h a r a c t e r i s t i c s , p a r t i c u l a r l y i n the d i s t r i b u t i o n and degree of exposure of surface glycoproteins and other 133 s t r u c t u r a l moieties, appear to be associated with the co n t r o l of c e l l d i f f e r e n t i a t i o n and p r o l i f e r a t i o n . A l t e r a t i o n s of c e l l surface charac-t e r i s t i c s are c l e a r l y associated both with malignant transformation and with antigen- or mitogen-induced transformation, as w e l l as with processes of c e l l aggregation and d i f f e r e n t i a t i o n (Duzgunes, 19 75; Nicolson, 19 75). The expression of theta and other antigens on c e l l surfaces has been r e l a t e d to the d i f f e r e n t i a t i o n and maturation of T-c e l l s (Hammerling, 19 71; Raff, 19 70,; James et_ a l . , 19 74), and a loss of some antigens present during f e t a l development i s associated with the d i f f e r e n t i a t i o n of various other c e l l types. A r e d i s t r i b u t i o n (capping) of receptor s i t e s on lymphocytes i s associated with the i n t e r a c t i o n of these c e l l s with antigens and mitogens. C e l l - t o - c e l l contact also i n i t i a t e s a r e d i s t r i b u t i o n of i n t r i n s i c membrane proteins and i t has been suggested that this may be r e l a t e d to the co n t r o l of c e l l p r o l i f e r a t i o n (Scott et a l . , 1973). Neoplastic transformation also appears to cause a change i n the proteins of the plasma membranes of the transformed c e l l s , uncovering d i s t i n c t chemical groupings, and a l t e r i n g the morphology and behaviour of the c e l l s , and a l t e r i n g the co n t r o l of c e l l p r o l i f e r a t i o n (Duzgunes, 19 75). I t i s easy to envisage how some a l t e r a t i o n i n the c e l l surface could i n t e r f e r e with the action of hormones such as er y t h r o p o i e t i n on the c e l l , perhaps by i n t e r f e r i n g with hormone binding. S i m i l a r l y , an a l t e r a t i o n i n the c e l l surface could a l t e r the i n t e r a c t i o n of receptor molecules with antigens and mitogens. A defect i n migration, too, might r e a d i l y be r e l a t e d to some change i n the surface constituents 134 of the c e l l s , such that the interaction of those cells with surfaces is altered and migration thereby inhibited. Relatively l i t t l e is known, however, about the control of c e l l surface topography, or about the precise role different surface charac-teristics play in various c e l l a c t i v i t i e s . For this reason, i t is d i f f i c u l t to speculate on precisely what the W gene may be doing (or not doing) to cause i t s various effects. It is not necessary, however, to postulate a direct effect of the W gene on specific hormone receptors or on specific antigen and mitogen receptors. The distribution of receptors over the c e l l surface, the degree to which they are exposed, and their proximity to other moieties on the c e l l surface, may also affect the functioning of those receptors. The density and d i s t r i -bution of other structures and other charged moieties over the c e l l surface may also influence receptor function. Any of these charac-te r i s t i c s may be under the control of the W locus. Not a l l cells are equally affected by the W gene. Primordial germ cells and prospective pigment cells seem to be the most profoundly affected since these cells neither migrate nor proliferate nor differen-tiate. Erythroid progenitor cells are perhaps next most severely affected, and leukocytic progenitor cells next, both populations being limited in their response to proliferative stimulation. Lymphocytes themselves are only very mildly affected, being marginally slower in migration and somewhat altered in reactivity to antigens. Many c e l l types, however, respond quite normally to their respective hormones, many appear to migrate normally or very nearly normally, and, indeed, the response 135 of T - c e l l s to stimulation appears to be enhanced by the W mutation. This d i f f e r e n t i a l a ction of the W locus suggests that the c h a r a c t e r i s -t i c s which are c o n t r o l l e d by the W gene are of s i g n i f i c a n c e i n the a c t i v i t i e s of only c e r t a i n types of c e l l s . D i f f e r e n t types of c e l l s are characterized by d i f f e r e n t surface features, and i t may be the p r e c i s e combination of these features or t h e i r i n t e r a c t i o n , rather than any s i n g l e surface character, which i s most s i g n i f i c a n t i n c e r t a i n c e l l functions. The primary e f f e c t of the W gene, therefore, may be the same i n a l l c e l l s , but the ultimate e f f e c t of the mutation on sur-face features may be more pronounced or more far-reaching i n some c e l l s than i n others, at l e a s t i n terms of i t s s i g n i f i c a n c e f o r c e r t a i n func-tions . The hypothesis that the W locus controls a c h a r a c t e r i s t i c of the c e l l surface i s the only hypothesis yet proposed which appears able to account for a l l the p l e i o t r o p i c e f f e c t s of t h i s gene. For t h i s reason alone i t i s an a t t r a c t i v e hypothesis. I t i s , however, a d i f f i c u l t hypothesis to t e s t , f o r i n v e s t i g a t i o n s of c e l l surfaces are severely l i m i t e d by t e c h n i c a l procedures. The demonstration that the W gene has a profound e f f e c t on B - c e l l s , however, may be u s e f u l i f studies of the surface features of W/WV-type c e l l s are to prove f e a s i b l e . Other a f f e c t e d c e l l types i n these mice, p a r t i c u l a r l y p r i m o r d i a l germ c e l l s and prospective pigment c e l l s , are not obtainable f o r i n v i t r o s tudies; there are simply no techniques f o r i s o l a t i n g these c e l l s from developing embryos. In a d d i t i o n , considerable controversy s t i l l e x i s t s over the i d e n t i t y 136 of hematopoietic stem c e l l s , and s p e c i f i c c e l l precursors such as ery-thropoietin-responsive c e l l s are not r e a d i l y i s o l a t e d from the v a r i e t y of c e l l s i n bone marrow. Lymphoid c e l l s , on the other hand, are r e a d i l y obtained i n large numbers from thymus, spleen, and lymph nodes, and pure B - c e l l populations are obtainable i n a v a r i e t y of ways. These c e l l s are, therefore, convenient c e l l s to use i n i n v e s t i g a t i o n s of the c e l l surface. The use of l e c t i n s to probe the features of c e l l surfaces i s one of the more popular and s u c c e s s f u l methods now a v a i l a b l e f o r c e l l surface studies, and the use of l e c t i n s f o r t h i s purpose has been de-s c r i b e d i n d e t a i l by Nicolson (1975). Determination of the number and d i s t r i b u t i o n of l e c t i n - b i n d i n g s i t e s on lymphocyte surfaces, and a study of t h e i r r e d i s t r i b u t i o n following s t i m u l a t i o n , using the tech- • niques described by Nicolson (19 75), might reveal differences between +/+ and W/WV c e l l s which could be r e l a t e d to the d i f f e r e n c e s i n trans-formation which these populations of c e l l s e x h i b i t . The r e l a t i o n s h i p between l e c t i n binding, receptor r e d i s t r i b u t i o n , and b i o l o g i c a l response i s not at a l l c l e a r : binding of l e c t i n s to lymphocytes, f o r example, does not always r e s u l t i n receptor r e d i s t r i b u t i o n or i n c e l l trans-formation. Nevertheless, any differences between +/+ and W/WV c e l l s i n t h e i r response to l e c t i n s might provide some s i g n i f i c a n t information about the surface features of these c e l l s . I t has been suggested that c e l l cytoplasmic s t r u c t u r e s , such as networks of microtubules and microfilaments located near the inner surface of the c e l l membrane, may c o n t r o l the m o t i l i t y and d i s t r i b u t i o n 137 of membrane proteins (Nicolson, 1973). I f t h i s i s true, the action of the W gene may be to i n t e r f e r e with the structure or function of microfilaments or microtubules, and thereby to a l t e r the e f f e c t which r e d i s t r i b u t i o n of membrane structures normally exerts on the c e l l . This hypothesis seems perhaps u n l i k e l y , since so many c e l l types i n W/Wv mice appear to function normally. Whether or not the W locus does a f f e c t c e l l cytoplasmic s t r u c t u r e s , however, could be determined by u l t r a s t r u c t u r a l studies of af f e c t e d c e l l types. In addition to probing c e l l surface receptors, t h e i r i n i t i a l d i s t r i b u t i o n , and t h e i r r e d i s t r i b u t i o n following l e c t i n binding, f u r t h e r v studies concerning immune reactions of W/W mice might help i n exploring v the c e l l surface hypothesis. The a b i l i t y of c e l l s from W/W mice to respond to B - c e l l mitogens and antigens other than LPS and SRBC might reveal that not a l l B - c e l l responses i n W/WV mice are d e f i c i e n t . Such a r e s u l t would'suggest that antibody production i t s e l f i s not impaired i n these mice, and would make the hypothesis of a general B - c e l l de-f i c i e n c y u n l i k e l y . The detection of normal responses to some antigens i n W/WV mice would not be in c o n s i s t e n t with the hypothesis that the defect i n W/WV c e l l s , when i t i s manifest, l i e s at or near the c e l l surface i n t e r a c t i o n . CONCLUSION The p o s s i b i l i t y that the W locus controls a c h a r a c t e r i s t i c of the c e l l surface which influences the i n t e r a c t i o n of c e l l s with environmental s t i m u l i i s an e x c i t i n g hypothesis, not only because i t can explain a l l the known e f f e c t s of the W mutation i n mice, but also because i t may provide a new system i n which to i n v e s t i g a t e the c o n t r o l of c e l l migration, p r o l i f e r a t i o n , and d i f f e r e n t i a t i o n . C e l l surface phenomena are c r i t i c a l components of both the migratory process and the induction of p r o l i f e r a t i o n and d i f f e r e n t i a t i o n i n many c e l l types. 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