Phagocytosis and MHC II antigen presentation by human γδ T cells Luísa de Jesus Saraiva Molecular Immunology Unit UCL Institute of Child Health London A thesis submitted for the degree of Doctor of Philosophy August 2014 1 Declaration I, Luísa de Jesus Saraiva, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 1 Abstract γδ T cells are a rare subset of T cells present in human blood and lymphoid tissues. Functionally, they stand at the interface of the innate and the acquired branch of the immune system as they express somatically rearranged antigen receptors but they also share many characteristics with NK cells. One of those is the ability to express CD16. CD16 is a cell-surface receptor that binds the Fc portion of IgG. In the isoform found on tissue macrophages and NK cells it is coupled to an intracellular signalling domain that allows it to initiate cellular processes leading to the internalisation of IgG-coated particles or cytotoxicity against IgG-coated cells. Therefore, it has a role in the clearance of IgG-coated complexes and the destruction of infected or malignant cells. In this thesis, I show that a population of γδ T cells present in human blood from healthy donors express the CD16 receptor and implicate a role for this receptor in phagocytosis of IgG-coated bacteria and synthetic beads coated with recombinant influenza M1 protein. These cells were subsequently shown to activate influenza-specific T cells through MHC II presentation. Importantly, neither αβ T cells nor NK cells were capable of significant phagocytosis. In addition, once CD11c+ cells had been removed from the NK fraction, neither αβ T cells or NK cells were capable of MHC II antigen presentation of influenza M1 antigen. The CD16+ γδ T cells displayed a phenotype of effector memory and late effector memory T cells suggesting a role for these cells in patrolling the blood and rapidly migrating to sites of inflammation for a combination of rapid effector and antigen presentation function. 3 Acknowledgments First and foremost I would like to thank my supervisor Dr Kenth Gustafsson for taking me on as a student and for his endless enthusiasm and open-minded approach to science. This project would not have been possible without his patience and encouragement and many helpful intellectual discussions. I would also like to thank my co-supervisor Dr Siobhan Burns for taking me on first as a research assistant and then as a PhD student and for actively training me to become technically competent and providing guidance on the direction of research. I would also like to acknowledge the extensive contribution of Dr Conrad Vink as an editor throughout the re-drafting process, whose constructive criticism greatly improved the standard of this document. I would like to thank Dr Yin Wu whose intellectual contributions made this project possible in the first place. In addition, I would like to acknowledge Dr Ayad Eddaoudi for technical assistance with flow cytometry and cell sorting and Dr Bertrand Vernais for technical assistance with confocal microscopy, Julien Record and Dr Dale Moulding for helping me with the granulocyte isolation, staining and microscopy, and Dr Giorgia Santilli for helping me with the NBT assays. I am also thankful to Flávio Saraiva for writing a randomising software to score microscopy data, to Dr Marlene Carmo for helping me with the chromium release assays, and to Dr Holly Stephenson for training me in the gentamicin protection assay. A warm thank you to all the blood donors that contributed to this work and to the many members of MIU who make it a good place to work in. In particular, thank you to Dr João Metelo and João Nunes for patiently training me during the time I spend working with them and for their warm hospitality. Thanks to Aris whose 6 am thesis starts whilst doing army duty provided a standard no subsequent PhD student can hope to match. Thank you to Maria Manunta for enduring tango lessons whilst trying to ignore her foot needed medical attention. Thank you to Fran and Gavin for being the best desk neighbours. Thank you to Mus for his unshakable drive. Thank you to Sarah for her endless optimism and energy. Thank you to Ida for her encouragement and for showing us you can have it all. In addition, thank you to the rest of the MIU crowd in the upstairs offices for boozy Friday pub outings and over-ambitious cycling missions. 4 Thank you to the friends that keep me grounded and remind me of other times, scattered they may be around the world. A special thank you to Ana Luísa in Stockholm and Lina in Zurich. Thank you for your moral support, advice and laughter. Thank you also to Miguel, Catarina, Diana, Daniela, João Nascimento, Marta and Rita who liven up going back home. Thank you to my mum for being such an inspiration throughout her life’s struggles, and for completing her own PhD with three demanding children at her back. Thank you to my dad for his independent thinking and integrity and his selfless love and kindness. Thank you to my brother Flávio and my sister Inês for believing that there is no such thing as too much education! To Conrad, thank you for keeping me sane and hopeful when times were rough and for making every day brighter. 5 List of Figures Figure 4.1 – Detection of phagocytosis of opsonised green fluorescent E. coli by γδ T cells using confocal microscopy ................................................................................................ 74 Figure 4.2 – Distinguishing intracellular E. coli from surface bound E. coli by confocal microscopy z-stacks .................................................................................................................... 75 Figure 4.3 – Examples of confocal microscopy z-stacks where the distinction between intracellular E. coli and surface bound E. coli is ambiguous ................................................... 76 Figure 4.4 – Discrimination between intra and extracellular E. coli associated with γδ T cells using dual fluorescence flow cytometry .......................................................................... 79 Figure 4.5 – Establishment of live GFP-expressing E. coli cultures as an alternative to lyophilised fluorescein-labelled E. coli for the study of phagocytosis in γδ T cells ............. 82 Figure 4.6 – Comparing phagocytosis of lyophilised fluorescein-E. coli and live GFP-E. coli by confocal microscopy ........................................................................................................ 83 Figure 4.7 – Internalisation of opsonised live E. coli by γδ T cells is detected in a standard gentamicin protection assay ....................................................................................................... 86 Figure 4.8 – Comparative quantification of phagocytosis of highly purified leukocyte fractions using blind-scored confocal microscopy images .................................................... 90 Figure 4.9 – A population of CD3+ γδ TCR+ cells in whole blood can phagocytose opsonised GFP-expressing E. coli ............................................................................................. 94 Figure 4.10 – Characteristics of phagocytosing and non-phagocytosing CD3+ γδ TCR+ cells in the whole blood phagocytosis assay ............................................................................ 97 Figure 4.11 – Phagocytosing CD3+ γδ TCR+ cells in whole blood produce reactive oxygen species .............................................................................................................................. 99 Figure 4.12 - Human blood γδ T cells include cells with unusual dendritic-like extensions ...................................................................................................................................................... 100 Figure 4.13 – Phagocytosing CD3+ γδ TCR+ cells in whole blood have unusual morphology ................................................................................................................................ 103 Figure 4.14 – Purity assessment of FACS sorted γδ T cells by flow cytometry ............... 107 Figure 4.15 – Purity of non-phagocytic and phagocytic FACS sorted γδ T cells ............. 108 Figure 4.16 – The CD3/ γδ TCR complex is not expressed in human neutrophils or eosinophils .................................................................................................................................. 112 Figure 4.17 – The γδ TCR is not expressed by polymorphonucleated cells ..................... 113 Figure 5.1 – Class II presentation of M1 influenza antigen by γδ T cells .......................... 125 Figure 5.2 – γδ T cells present significantly more M1 influenza antigen than other lymphocytes ................................................................................................................................ 128 Figure 5.3 – Antigen presentation was seen in some wells of NK cells directly isolated with CD56 beads but not when NK cells were isolated by depletion of non-NK cell types ...................................................................................................................................................... 132 6 Figure 5.4 – Comparison of antigen presentation by freshly isolated γδ T cells and γδ T cells which have undergone γδ T APC maturation .............................................................. 135 Figure 5.5 – CD16, class II antigen presentation