Study of the inheritance of a polymorphic human trophoblast antigen and its equine equivalent A thesis submitted by Abbas Rezai in partial fulfilment of the requirementsfor the degreeof Doctor of Philosophy in the University of London October 1995 Department of Immunopathology St Mary's Hospital Medical School 1 IN THE NAME OF ALLAH, THE BENEFICENT, THE MERCIFUL To my family 2 ACKNOWLEDGEMENTS My main thanks and deep appreciation are due to Professor Mowbray for his invaluableadvice, help, encouragementand helpful criticism throughout the duration of this research. Without his guidance and stimulation this study would not have been completed. I would like to thank Dr J Underwood for her continuing helpful comments, correction and proof reading. I would like to thank Professor Allen and S Mathias for their help in collection of the horse placentaeand collaborative studies. This thesiswould not have materialisedwithout their help. I would also thank to my friend, R Jalali, for all of his kindness over the years. I wish to thanks Professor's Mowbray family and all of those who volunteered to participate in some of this studies necessaryfor the collection of the blood samples. My thanks is also due to the Labour Ward Staff of St Mary's hospital for their help in collection human placentae. I would like to extend my gratitude to the Ministry of Health and Medical Education of Iran for it's financial support and scholarship. I would also like to thank my parents for their patience. Finally my special tributes are to my wife, Mahnaz, for her patience and moral support which allowed me to focus on my study, despite her sickness. 3 ABSTRACT It has been shown that a polymorphic antigen, R80K, is expressed on the maternal surface of the human placenta. The antigen is covered by specific maternal IgG. A similar trophoblast antigen is also found on horse placenta, it appears to be analogous to human R80K. The antigen is covered with specific IgG antibody and following trypsin digestion it behavesin the same way as the human R80K. A stallion will cover as many as 30 mares in one breeding season,thus each foal has a common father but different mother. This facilitated the study of distribution of the R80K antigen in the progeny. I have studied the inheritance of the paternal allotypes of this protein in order to investigatethe distribution of allotypes in sibships. The R80K trophoblast antigen which immunisesthemother is of paternal origin. The inheritance of paternal genes thus in part determines whether the mother can respond to a particular antigen. It has been suggested that antibody to trophoblast antigens may protect from the immune maternattack and subsequent abortion. Women with repeated early pregnancy failure usually do not have detectable antibody to MHC alloantigens; I have found that they do not usually exhibit antibodies to the R80K allotype either. The commonestpattern of recurrent abortion is the loss of all pregnanciesor all but the first. Since the condition is rare and women would be expected to make antibody to nearly all allotypes, it would be difficult to equate lack of antibody to R80K as responsiblefor recurrent abortion, as random inheritance of either of the two paternal allotypes would produce only a 50% loss. A possible explanation might be very unequal transmission of the two paternal R80K allotypes. Thus, instead of a high frequency of losing half the pregnancies,they might show loss of all pregnanciesif they all had only the one allotype to which the mother could not make protective antibody. IgG antibodies were eluted from the human placenta, 3 families of horses and some unrelated horse controls by acid. The reactions between the eluted IgG antibodies and the antigen on the 4 remaining acid treated microvesicleswere measuredand used to identify the antigenic groupings in the families. The results can be summarisedas follows: Equine antibodies eluted from placental microvesicles with acid did not react with human acidified microvesiclesor peripheral blood cellsnor did human antibodiesreact with either horses cells or acidified horse microvesicles. The eluted antibody always reacted with its own antigen and with the acidified microvesiclesof all half siblings in its family. Occasionally the same antigen was detected on unrelated placentae. I conclude that there is highly unequaltransmission of the two paternalR80K allelesin horses. 5 CONTENTS PAGE No 1. Introduction 12 1.1 Mendel's Modes of Inheritance. 1.1.1 Autosomal dominant inheritance. 1.1.2 Autosomal recessiveinheritance. 1.1.3 Sex linked inheritance. 1.1.3.1 Y-linked (Holanderic) inheritance. 1.1.3.2 X-linked recessiveinheritance. 1.1.3.3 X-linked dominant inheritance. 1.2 Transmissionratio distortion. 1.2.1 Transmissionratio distortion of the t- haplotype genesin mice. 1.2.2 Genomic Imprinting. 1.2.2.1 MonoaUelic expression. 1.2.2.2 Monoallelic changesin the genome. 1.2.2.3 DNA Methylation as a mechanismfor imprinting. 1.3 Placentation. 1.3.1 Comparative placentation in Equids and humans. 1.3.2 Placentation in man. 1.3.2.1 Cytotrophoblast and Syncytiotrophoblast. 1.3.2.2 Chorion and Villi. 1.3.3 Placentationin horses. 1.4 Expression of antigensby Trophoblast. 1.4.1 Presenceof an 80kDa antigen on microvillus plasmamembrane. 1.4.2 MHC ClassI and II. 1.4.3 Trophoblast lymphocyte cross reactive antigen. 1.4.4 Placental alkaline phosphatase. 1.4.5 Fc receptors ligands for IgG in the human placenta. 1.5 AUoantibodiesin the Sera of PregnantWomen. 1.5.1 HLA antibodies. 1.5.2 Mixed lymphocyte culture (MLC) blocking antibodies. 1.5.3 Anti-idiotypic antibodies. 1.5.4 Anti-Trophoblast antibodies. 1.6 The aim of my study. 2. Materials and methods. 52 2.1 Preparation of microvesicles. 2.1.1 Equine placentae. 2.1.2 Human placentae. 6 2.2 Elution of IgG antibody bound to microvesicles. 2.3 Isolation of IgG from human sera. 2.4 Single radial immunodiffusion (SRID). 2.5 Measurementof equine IgG concentration. 2.5.1 Standard curve. 2.5.2 Concentration of samples. 2.6 Lymphocyte preparation. 2.7 Preparation of B-lymphocyte suspension. 2.7.1 Preparation of 1% sheepred blood cells. 2.7.2 Separationof T and B-lymphocytes. 2.8 Preparation of cells by dextran sedimentation. 2.9 Monocyte preparation. 2.10 Staining of peripheral blood cells. 2.11 Collection of sera. 2.12 Radioiodination of proteins. 2.13 Reaction of acidified vesicleswith acid eluted IgG antibody. 2.13.1 Equine vesicles. 2.13.2 Human placentalmicrovesicles. 2.14 Methods for detection of the R80K antigen on the surfaceof peripheral cells. 2.14.1 Binding of acid eluted IgG antibody to peripheral blood cells. 2.14.2 Detection of antibody using FACS. 2.14.3 Complementdependent cytotoxic antibody test. 2.15 Detection of anti R80K antibody in sera of pregnant women and women with recurrent spontaneousabortion. 2.16 Trypsin digestion of microvesicles. 2.17 SDS PAGE. 2.17.1 Staining and destainingof gels. 2.18 Purification of proteins. 2.18.1 Gel filtration chromatography. 2.18.2 Purification of eluted IgG antiboy from human placentaeby affinity chromatography. 7 2.19 Statistical methods. 3. Results. 69 3.1. Studies of the IgG bound to syncytiotrophoblastmicrovesicles. 3.1.1 Acid elution of antibody from human microvesicles. 3.1.2 Acid elution of antibody from horse placentalmicrovesicles. 3.1.3 Reaction of human eluted antibody with acid treated microvesicles. 3.1.4 Determination of optimal conditions for the detection of binding of cells and antibody. 3.1.5 Eluted human antibody reactswith the father's cells rather than a third party. 3.1.6 Microscopy of dextran and Lymphoprep preparedhuman peripheralblood cells. 3.1.7 Reaction of human antibody with cultured lymphoblastoidB-lymphocytes cell lines homozygousfor IHLA. 3.1.8 Reaction of equine eluted antibody with acid treated vesicles. 3.1.9 Reaction of acid eluted antibody with different preparationsof peripheralblood cells. 3.1.10 Acid eluted IgG antibody is speciesspecific. 3.2 Studies of the antigen detectedwith eluted antibody. 3.2.1 Reaction of the 50KDa fragment with acid eluted antibodies. 3.2.2 Trypsin digestion of horse vesicles. 3.3 Cross reactions of antigen and antibody betweenindividuals of the samespecies. 3.3.1 Cross reactions of humantrophoblast by binding assay. 3.3.2 Human cross reaction of Lymphoprep and dextran prepared cells by a binding assay. 3.4. Familial distribution of antigen allotypes. 3.4.1 Transmissionof a paternal antigen in a human family. 3.4.2 Typing of Nearco family membersusing acid eluted antibodiesand acid treated vesiclesof individual placentae. 3.4.3 Cross reaction within the family shown by eluted antibody to horse PBC. 3.4.4 Distribution of equine allotypes in the family of Nearco. 3.5. Antibodies to paternal allotypes in maternal sera. 3.5.1 Detection of antibodies in sera of women with normal pregnancy by binding to paternal PBC. 3.5.2 Detection of antibodies in sera of women with repeated abortion. 3.5.3. Detection of antibodiesto husbands'PBC in sera of immunisedwomen. 4. Discussion 106 5. Bibliography 122 6. Appendix A 141 7. Appendix B 143 8 List of illustrations. Figures Page No. 1.16 2.19 3.71 4.73 5.74 6.80 7.81 8.87 9.91 10.99 9 List of Illustrations. Tables Page No. I. 28 II. 31 III. 72 IV. 75 V. 78 VI. 79 VII. 82 VIII. 83 IX. 84 X. 85 M. 88 XII. 89 XIII. 92 XIV. 94 Xv. 95 XVI. 97 x vii. 101 XVIII. 103 M. 104 XX 105 Plates I, 70 II 76 III 77 10 List of abbreviations used A Abortion Ab Antibody Ae Acid eluted antibody. AMPS Ammonium persulphate APCA Antipaternal cytotoxic antibody.