IMMUNOPHENOTYPING OF LYMPHOCYTES IN HEALTHY AND IMMUNODEFICIENT CHILDREN Vries, Esther de Immunophenotyping of lymphocytes in healthy and immunodeficient children I Esther de Vries. - Rotterdam: Erasmus Universiteit Rotterdam, Afdeling Immunologie. - ill. Proefschrift Rotterdam. - Met lit. opg. ISBN 90-73436-47-8 Trefwoorden: Immunofenotypering, immunodeficientie, Iymfocyten, kinderen. Key words: Immunophenotyping, immunodeficiency, lymphocytes, children. No part of this thesis may be reproduced or transmitted in any form by any means, electronic or mechanical, including photocopying, recording or IMMUNOLOGIE any information storage and retrieval system, ROTTERDAM without permission in writing from the publisher (Department of Immunology, E. de Vries, Erasmus University Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands). IMMUNOPHENOTYPING OF LYMPHOCYTES IN HEALTHY AND IMMUNODEFICIENT CHILDREN IMMUNOFENOTYPERING BIJ GEZONDE KINDEREN EN KINDEREN MET EEN AFWEERSTOORNIS PROEFSCHRIFT ter verkrijging van de graad van doctor aan de Erasmus Universiteit Rotterdam op gezag van de rector magnificus Prof. dr. P.W.C. Akkermans MA en volgens besluit van het College voor Promoties. De open bare verdediging zal plaatsvinden op woensdag 28 april 1999 om 15.45 uur door Esther de Vries geboren te Amsterdam. PROMOTIECOMMISSIE: Promotoren: Prof. dr. J.J.M. van Dongen Prof. dr. J.M.J.J. Vossen Overige leden: Prof. dr. R. de Groot Prof. dr. R. Benner Prof. dr. H.J. Neijens Dr. M.J.D. van Tol Het uitgeven van dit proefschrift werd mede mogelijk gemaakt door: Abbott B.V., Amgen BV., Boehringer Ingelheim BV., Glaxo Wellcome BV., Merck Sharp & Dohme B.V., Pfizer B.V., Astra Pharmaceutica BV., Friesland Nutrition en Alk Benelux BV. Omslagontwerp: Sascha Jansz, 10 jaar. Dit proefschrift werd bewerkt binnen de vakgroep Immunologie van de Faculteit der Geneeskunde en Gezondheidswetenschappen, Erasmus Universiteit Rotterdam/Academisch Ziekenhuis Rotterdam, de vakgroep Kindergeneeskunde van de Faculteit der Geneeskunde, Leids Universitair Medisch Centrum, en de vakgroep Kindergeneeskunde van het Sophia Kinderziekenhuis/Academisch Ziekenhuis Rotterdam. Het proefschrift werd gedrukt door Haveka B.V. te Alblasserdam. A/s schepen e/kaar mel unverfroren vader/andsliefde de grond inboren van de indonesische archipel a/s raketten sneller dan hel ge/uid afgevuurd aile kanlen uit ons ruimschools voorzien van kippevel is er moed voor nodig om ler wereld Ie komen - maar zie, de zon schijnl door de bomen en de meisjes hebben zich mooi gemaakt. is hel geen heer/ijke dag vandaag? de feeen om je wieg zullen goed voor je zorgen E s I her - wees welkom. Goedemorgen! januari 1962 Wim Wi/mink Aan mijn ouders, Arjan, Sascha en Robin 7 IMMUNOPHENOTYPING OF LYMPHOCYTES IN HEALTHY AND IMMUNODEFICIENT CHILDREN CONTENTS PART I Technical aspects of immunophenotyping in children Chapter 1: Introduction 11 Chapter 2: Flow cytometric immunophenotyping of lymphocytes 17 Chapter 3: Correction for erythroid cell contamination in 25 microassay for immunophenotyping of neonatal lymphocytes Arch Dis Child, 1999, in press PART II Lymphocyte subpopulatlons in healthy children Chapter 4: Analyzing the developing lymphocyte system of neonates 37 and infants Eur J Pediatr, 1999, in press Chapter 5: Neonatal blood lymphocyte subpopulations: 51 a different perspective when using absolute counts Submitted Chapter 6: Longitudinal follow-up of blood lymphocyte subpopulations 65 from birth to one year of age J Pediatr 1998;133:586-588 Chapter 7: Longitudinal survey of lymphocyte subpopulations in the 71 first year of life Submitted 8 PART III Lymphocyte subpopulations in Immunodeficient children Chapter 8: Immunophenotyping in the diagnosis and 95 follow-up of immunodeficient children Submitted Chapter 9: Reconstitution of lymphocyte subpopulations after 109 pediatric bone marrow transplantation Submitted Chapter 10: Identification of an unusual FcyReceptor Ilia (CD16) on 127 natural killer cells in a patient with recurrent infections Blood 1996;88:3022-3027 Chapter 11: TCRall+ T-Iymphocytopenia in two siblings 139 Submitted Chapter 12: The 782C-n (T254M) XHIM mutation: lack of a tight 147 phenotype-genotype relationship Submitted Chapter 13: A girl with agammaglobulinemia and absent blood 153 B-Iymphocytes PART IV Discussion and summary Chapter 14: General discussion 163 Summary 171 Samenvatting 177 Dankwoord 179 Curriculum vitae 183 PART I TECHNICAL ASPECTS OF IMMUNOPHENOTYPING IN CHILDREN I I I I I I I I 11 CHAPTER 1 INTRODUCTION 13 INTRODUCTION This thesis describes the use of immunophenotyping of lymphocytes in healthy as well as immunodeficient children. Part I describes the applied techniques (Chapters 2 and 3). The experimental work in healthy children is described in Part II (Chapters 4-7) and Part III describes the experimental work in immunodeficient children (Chapters 8-13). A summary and discussion of the work is given in Part IV. Chapter 2 shortly describes some technical aspects of flow cytometric immunophenotyping of lymphocytes. The advantages of using the lysed whole blood technique with directly conjugated monoclonal antibodies are discussed. In Chapter 3 a microassay for immunophenotyping of neonatal and infant lymphocytes is described. This microassay was developed for the study of healthy children during their first year of life, to enable detailed immunophenotyping in a small volume of blood (0.5 to 1.0 ml). In neonatal cord blood samples, contamination of the flow cytometric "Iympho-gate" wHh erythroid cells leads to underestimation of lymphocyte subpopulations. This erythroid contamination can be easily identified with the GpNCD71/CD45 triple immunostaining, which allows correction for the presence of erythroid cells wHhin the "Iympho-gate". Chapter 4 constitutes the introduction to the immunophenotyping stUdies in healthy children. Problems and pHfalis encountered in the study of the developing pediatric lymphocyte system are discussed, and literature data on the current knowledge of lymphocyte development in neonates and infants are summarized. The age-related differences in lymphocyte subpopulations and function imply that the available adult reference values cannot be used in children. In Chapter 5 the actual sizes (Le. the absolute counts) of the "naive" and "mature" blood lymphocyte subpopulations are compared between preterm neonates, term neonates, and adults. Unexpectedly, "mature" blood lymphocyte subpopulations appeared to be present in comparable absolute counts in term neonates and adults. However, "naive" lymphocyte subsets occurred in essentially higher absolute counts in term neonates, suggesting that a large pool of "naive" (untriggered) cells is standby in neonates, ready for partiCipation in primary immune responses. This pool of "naive" lymphocytes was not so large in some preterm neonates, which may partly explain their higher susceptibility to infections. Chapter 6 presents the longitudinal follow-up study of T-Iymphocytes, 8- lymphocytes, and NK-cells in ten healthy children from birth to one year of age. T­ and B-Iymphocytes increased at one and six weeks of age, respectively, whereas NK-cells showed a sharp decline directly after birth. These data show that the changes in size of the lymphocyte subpopulations that are found in cross-sectional studies are confirmed in longitudinal studies of individual children. 14 CHAPTER 1 In Chapter 7 the longitudinal follow-up of several subpopulations of T­ lymphocytes, B-Iymphocytes, and NK-cells during the first year of life is shown. Unexpectedly, the CD45RO+ T -lymphocyte count did not change during the first year of life, whereas the CD45RA+ T -lymphocyte count increased markedly, providing a large pool of cells for participation in primary immune responses. CD38 (T- and B-Iymphocytes) and CD1c and CD5 (B-Iymphocytes) are discussed as possible markers of peripheral immaturity. Finally, NK-cell subpopulations and their possible role before and after birth are discussed. Chapter 8 gives an introduction to the immunophenotyping studies in immunodeficient children. A stepwise protocol for the diagnosiS of immunodeficiency is presented, including an immunophenotyping protocol for clinical use. The role and interpretation of immunophenotyping results in the diagnostic process are discussed. In Chapter 9 the use of immunophenotyping in studying the slow reconstitution of lymphocyte subpopulations after pediatric bone marrow transplantation is illustrated. Especially the CD4+ helper T-Iymphocytes recovered slowly and reached the p, of age-matched reference values at a late stage (> 6 months posttransplant). The unexpected acceleration of the reconstitution of CD4+ helper T -lymphocytes in patients with CMV-reactivation is discussed. The posttransplant development of the absolute counts of CD45RA+, CD45RO+, and CD7- T­ lymphocyte counts are discussed in relation to normal ontogeny and the possibility of peripheral expansion of previously primed donor-derived "memory" T­ lymphocytes versus newly processed T-Iymphocytes via T-Iymphopoiesis in the thymus. In Chapter 10 the use of immunophenotyping is described for the identification of an unusual F,yReceptor lila (CD16) on NK-cells in a patient with recurrent infections. Loss of the epitope for the CD16 (B73.1) monoclonal antibody led to the identification of F,yRIIIA-48H/H homozygosity. The combined use of CD16 and CD56 monoclonal antibodies labeled with the same fluorescent dye, as is often applied in routine immunophenotyping procedures, will leave these