T cells and immunity to the asexual blood stages of Plasmodium chabaudi chabaudi CB by Kathleen Emma Mathers BSc (University of Reading) Wellcome Laboratories for Experimental Parasitology Department of Zoology University of Glasgow A thesis submitted for the degree of Doctor of Philosophy in the University of Glasgow © K.E. Mathers, June 1994 ProQuest Number: 13834132 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 13834132 Published by ProQuest LLC(2019). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 lLh> 'j'jOit GLASGOW UNIVERSITY Cfl' LIBRARY FOR MY PARENTS TO WHOM I OWE EVERYTHING SCIENCE MOVES BUT SLOWLY SLOWLY, CREEPING ON FROM POINT TO POINT ALFRED, LORD TENNYSON GLASGOW UNIVERSITY LIBRARY TABLE OF CONTENTS CHAPTER TITLE PAGE NO. Acknowledgements ii Declaration iv Abbreviations v Summary viii 1 General Introduction 1 2 Materials and Methods 47 3 Selective depletion of T cell subsets in vivo 68 4 Adoptive transfer of immunity with splenic lymphocytes 79 5 Passive transfer of immunity and analysis of recrudescent 110 parasite populations 6 The role of nitric oxide during the course of infection 124 7 In vitro propagation of malaria specific T cell lines 140 8 General Discussion 154 Appendix 162 References 165 ACKNOWLEDGEMENTS I would like to thank Professor R.S. Phillips for his advice and guidance during my period of research at W.L.E.P. and for his constructive criticism and discussion during the preparation of this thesis. I am also grateful to Andrew Taylor- Robinson and Lisa Brannan for all their initial help in showing me the ropes and teaching me many of the techniques employed in this thesis. Extra special thanks must go to Fiona McMonagle and David McLaughlin for their excellent and invaluable technical assistance throughout the course of my experimental work and especially with the production of anti-CD4 and anti-CD8 monoclonal antibodies. Fiona and David have both bome my endless questions and pleas for help with patience, willingness to lend a hand and constant friendship. Thanks also to Fiona for instruction in the Apple Mac, for persuading me to take some exercise and for allowing pervading vinegar smells in her kitchen during our chutney making sessions. Colin Chapman also deserves big thanks not only for the care of my animals at weekends but also for his friendship, many chats on Friday night drinking sessions and for being such an excellent ceilidh partner. The technical help of Anne McIntosh, Christine Hamilton and Sandra Terry is also very much appreciated. For provision of clean and sterile glassware I thank Isobel, Liz and Anna. I am very grateful to Diana McKechnie for access to the 60Co source and for her cheery welcome on my visits to Veterinary Physiology. Thanks also to Kathryn Brown for giving me some J774 cells; David Walliker for providing CB parasites while mine failed to grow; Alison Severn for carrying out the initial nitric oxide results; Kate O'Donnell and Jacqui Doyle for instruction in nitrite and nitrate analysis and for letting me loose on the chemiluminescence analyser; Julie Osborne, Sarah Hunter and Eileen Devaney for much help with ELISAs and Liz Denton for scanning some pictures for this thesis. I must also thank Hal Thompson and his colleagues at Veterinary Pathology for performing numerous post mortems and for their interest in the resulting findings. I am indebted to Malcolm Kennedy, Eileen Devaney and Paul Hagan for advice and useful discussions. Philippe Neuville and Joyce Moore gave up much of their time for long chats and helpful advice, often over a pint (usually not bought by me!) - thank you. Special thanks to Eileen, Dave and Ewen for such an enjoyable source of much needed pocket money. For making my time at W.L.E.P such a varied but mostly fun and interesting one I thank everyone in the lab. over the years - Fiona, David, Anne, Jacqui, Andrew, Lisa, Amanda, Philippe (the IOU grows!), Stephen, Malcolm, Joyce, Heather, Collette, Karen, Lesley, Yu, Liz, Anna, Isobel, Christine, Sandra, Colin, Graham and Valentina. To all my assorted friends since my arrival in Glasgow: Elaine, Karen, Ros, Roddy, Toby, Graham, Alan, John, Alan, Kathleen, Kate, Jayne, Richard and Fiona, Tony (we'll crack the Pride of Erin waltz sometime soon), Frank (for pulling me out of a very large hole), Ian, Callum and Walter, Christine and Brenda, thank you all for putting up with me, making me feel at home and for joining me in frequent pints. Many many thanks to Elaine. For sharing turmoils, and a flat for three years, for constantly feeding and watering me, for nagging me to stop working, for trips to the cinema, for shopping sprees, for a shoulder to cry on, for many parties and drinking sessions and for nursing me through glandular fever and Beijing 'flu, - thank you, I owe you a big one! Finally, thanks to all my family and friends at home especially Sheelagh and Moira for long telephone conversations, advice and drunken New Years! Thanks are not enough for Mummy and Daddy for their constant love, encouragement, enthusiasm and support for all my endeavours. I dedicate this thesis to you. This work described in this thesis was performed whilst I was the recipient of a postgraduate studentship from the Medical Research Council, to whom I am extremely grateful. DECLARATION I declare that this thesis is my own composition and that the research described herein was performed entirely by myself except where expressly stated. Kathleen E. Mathers June 1994 iv ABBREVIATIONS Ab Antibody Ag Antigen APC Antigen presenting cell ATS Anti-thymocyte serum B cell B lymphocyte BCG Bacille Calmette Gu&rin BP Blood passage BSA Bovine serum albumin CD Cluster designation Ci Curie(s) CM Cerebral Malaria CMI Cell-mediated immunity Con A Concanavilin A cpm Counts (of radioactivity) per minute CSP Circumsporozoite protein d day DDT Dichloro diphenyl trichloroethane DNA Deoxyribonucleic acid DPI Days post infection EBV Epstein Barr virus ELISA Enzyme-linked immunosorbent assay Fab Antigen-binding fragment of immunoglobulin Fc Crystalline fragment of immunoglobulin FCS Foetal calf serum FTTC Fluorescein isothiocyanate g Acceleration in the earth's gravitational field g Gramme(s) G Gauge Gy Gray(s) 3H-T Tritiated thymidine H-2 Mouse major histocompatibility complex Hb Haemoglobin HIV Human Immunodeficiency Virus HLA Human histocompatibility leucocyte antigens hr(s) Hour(s) ICAM-1 Intercellular adhesion molecule-1 IFA Indirect fluorescent antibody IFAT Indirect fluorescent antibody test IFN-y Interferon-gamma Ig Immunoglobulin n Interleukin i.p. Intraperitoneally i.u. International unit(s) i.v. Intravenously kDa Kilodalton(s) 1 Litre(s) L-NMMA L-NG-monomethyl arginine LPS Lipopolysaccharide M Molar mAb Monoclonal antibody MHC Major histocompatibility complex mg Milligramme(s) Pg Microgramme(s) min Minute(s) ml Millilitre(s) pi Microlitre(s) mm Millimetre(s) |im Micrometre(s) pM Micromolar MSP Merozoite surface protein MW Molecular weight ng Nanogramme(s) NK cell Natural Killer cell nm Nanometre(s) nmol Nanomole NMS Normal mouse serum NO Nitric oxide N 02' Nitrite anion n o 3* Nitrate anion nRBC Normal/uninfected red blood cell NRS Normal rat serum o 2- Superoxide anion OH- Hydroxyl radical PABA Para-aminobenzoic acid PBS Phosphate buffered saline PBL Peripheral blood lymphocytes Pf EMP P. falciparum erythrocyte membrane protein PHA Phytohaemagglutinin PMN Peripheral blood mononuclear cells pRBC Parasitised/infected red blood cell RBC Red blood cell RNI Reactive nitrogen intermediates ROI Reactive oxygen intermediates RT Room temperature s Second(s) S.D. Standard deviation SICA Schizont-infected cell agglutination test S/N Supernatant T cell T lymphocyte Tc T cytotoxic lymphocyte Th T helper lymphocyte TNF Tumour necrosis factor TNS Tumour necrosis serum Ts T suppressor lymphocyte u.v. Ultraviolet v/v Volume per volume WBC White blood cell WEP Wellcome Experimental Parasitology (prefix used to describe numbered batches of stabilate) WHO World Health Organisation w/v Weight per volume > Greater than < Less than % Percentage point(s) SUMMARY It is now clear that the major protective immune mechanisms to the erythrocytic stages of Plasmodium chabaudi chabaudi AS require the presence of CD4+ T lymphocytes. Both ThI and Th2 malaria-specific, cloned T cell lines can protect immunocompromised mice upon adoptive transfer. Infection with P. c. adamiy however, another subspecies of P. chabaudi, is thought to be resolved solely by antibody-independent mechanisms. In different host/parasite combinations, therefore, CD4-bearing lymphocytes mediate cellular protective functions and/or act as helper cells in protective antibody production. In addition, one report suggests that infection with P. c. chabaudi CB is controlled in an antibody-independent manner. Experiments were performed, therefore, to define further the role of T cells in immunity to rodent malaria using the CB strain of P. c. chabaudi in NIH mice. Although this strain is a virulent but usually resolving mouse malaria, it is often necessary to treat mice at the peak parasitaemia with subcurative doses of chloroquine. Such treatment, therefore, also allowed the effect of chemotherapy on the development of an immune response to be investigated. Using the P. c. chabaudi CB/NIH mouse system, selective depletion of T cell subsets by treatment with rat monoclonal antibodies to murine CD4 and CD8 determinants revealed that the CD4-bearing population of lymphocytes is critically required for protection against erythrocytic infection.