REGULATION OF T CELL CLONAL EXPANSION CHUA REN JIE ISAAC B.Sc (Hons), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY YONG LOO LIN SCHOOL OF MEDICINE DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2012   Acknowledgements This thesis embodies difficult times where progress seemed elusive and problems are never far At the same time, it also embodies the invaluable experience and techniques I’ve picked up along the way that would be useful in scientific research but the soft skills that will be useful through my journey in life This journey of doing a PHD gave me the first-hand experience that in life, ones accomplishments are not solely based on one’s hard work and effort but with the help and work of many people who has contributed in this thesis one way or other whose guidance, support for which I am indebted to and the wonderful people in my life that made this a part of this arduous journey as more bearable one Prof Kemeny: You have my sincere heartfelt gratitude for giving me the opportunity to my PHD in your lab and for allowing me to have creative reign to come up with my own project that is totally not our lab’s expertise I agree with you during our conversation that this made the progress of my project more difficult, but I have learned so much more from this experience as a person, not just in the area of science I also want to thank you for not just being a supportive supervisor, not just giving up your precious Saturdays off to meet me but as a life-mentor I’ve learned so many valuable life lessons from you during our supervision sessions You have such a big heart for people Dr Kaldis: Thank you for agreeing to be my co-supervisor for this project and your valuable advice from the perspective of a non-immunologist which made this thesis more “complete” Thank you for your generosity in providing me with the knockout mice and advice for my cell cycle experiments Dr Kaldis’ lab: I would especially like to thank a PHD student, Shuhui who has been very helpful with helping me upkeeping the p27KO mice that I use and for taking time to teach me some of the experimental techniques used in this thesis as well as the help render me in one way or another Vithya, the animal technician who has helped me with collecting the mice Prof Kemeny’s lab: You guys have been a wonderful bunch of people that I enjoy spending my time with both inside and outside the lab It really amazing how we’ve become from just lab colleagues to good friends whom I can go out for badminton, table tennis, steamboat buffets, KTV, beach outings, photography outings and the list goes on It is also wonderful to know that all of you are multi-talented, not just doing science but in many other areas “unscientific” as well I hope that we will continue to keep in touch and continue to organize such activities to together Dr Chris Yang: Chris, thanks for being such a great help and being so patient to teach me the molecular techniques that I’ve been using I really admire that you are always so positive and optimistic I’m sure you will continue to shine and be successful wherever you go You have also taught me a lot about economics and the financial system Shuzhen: Thanks for being “so brave” to actually let me take your pre-wedding photos for you Your confidence in my photography skill is surprising I wish you happiness in your newly “minted” marriage and all the best in your future endeavours Yafang: It is nice to have gotten to know both you and Shuzhen these years You and Shuzhen are really good in what you and extremely competent working with the mice, it is like second nature It is amazing I’m looking forward to shoot your couple portraits soon! Kenneth: You are always selfless and helpful, at times putting others in front of yourself, always giving me help whenever you can, it is much appreciated I see your wit and enjoy your straight-faced sarcasms too! (as long as I’m not at the receiving end…) Moyar: Though may sometimes be too forward and pushy, I know you are a nice person at heart and thanks for always   organising the “eating” outings I really enjoyed them And you’re a good hair and makeup artist by the way! Sophie, Nayana and Alan: Though, I may not have known you guys as long as the rest, you have been a very fun and nice bunch of people to be with and thanks for the help you have rendered me (whether in teaching me certain mouse-handling tips & tricks or suggestions) I really take them to heart Benson: Undoubtedly the most hardworking person in our lab Without handling all our orders, taking care of the mice colonies, I’m sure none of our progress would have accomplished as much, if not for your support Thanks Benson! Fei Chuin and Dr Hutchinson: Thanks for all the help with the cell sorting and teaching me the proper way of analzying flow data I’ve benefitted a lot from your knowledge and experience Fei Chuin, I really love your sunny disposition, stay cheerful and happy even though now you have two kids to look after, ok? Dr Paul: Thank you for your suggestions and advice how I could approach this project, it has given me a new way of looking at a problem My wife, Yuemin: Thank you for putting up with me doing my PHD for these years, especially when I had to spend my nights in the lab doing time course experiments or accompanying me to the lab on weekends You have been very understanding and gracious I know and have seen how tough and busy your work is as well but you have been very accommodating I owe you a lot Hopefully, now I can depart from such an irregular work schedule in future =p   Contents Chapter - Introduction 1.1 Overview of Immunology 1.2 Cellular components of the innate immune system 1.3 Humoral components of the innate immune system 1.4 Cellular components of the adaptive immune system T cells and T cell development Th1/Th2 paradigm 10 Transcription factors in Th1/Th2 differentiation 10 Th17 cells 11 Natural and inducible regulatory T cells 12 Follicular T helper cells (Tfh) 13 1.5 Why study T cell clonal expansion? 13 1.6 T cell expansion rate and burst size 19 1.7 CD8+ and CD4+ T cells are intrinsically different 21 1.9 Cyclins and Cyclin-dependent kinases (CDKs) 26 1.10 Cyclin –dependent kinase inhibitors (CKIs) 26 p18ink4c 27 p19ink4d 28 p21cip1 28 p57kip2 28 p27kip1 29 1.11 Kinetics of p27kip1 levels in wild-type CD8+ and CD4+ 30 1.12 CD28 and cytokine signalling linked to p27kip1 down-regulation 31 1.13 Aims of the study 33 1.14 Specific Aims 34 Chapter - Materials and Methods 35 2.1 Materials, media and buffers 35 2.1.1 PBS buffer 35 2.1.2 MACS buffer 35 2.1.3 FACS buffer 36 2.1.4 Annexin-V binding buffer 36 i  2.1.5 Optiprep density centrifugation media for splenic DC isolation 36 2.1.6 Digestion buffer for spleen DC isolation 37 2.1.7 Complete medium for cell culture 37 2.1.8 DPBS buffer 38 2.1.9 Ethanol fixation solution 38 2.1.10 PI staining solution 38 2.1.11 4x Upper Tris 39 2.1.12 4x Lower Tris 39 2.1.13 10% APS 39 2.1.14 SDS running buffer 40 2.1.15 Western blot transfer buffer 40 2.1.16 PBST buffer & Western blot blocking buffer 40 2.1.17 List of antibodies used 41 2.2 Animals 44 2.2.1 Mice 44 2.3 Methods 45 2.3.1 Isolation of T lymphocytes 45 2.3.2 Purification of CD8+ and CD4+ T lymphocytes 47 2.3.3 Analysis of surface markers using Fluorescence-Activated Cell Sorter (FACS) 48 2.3.4 Isolation of CD11c+ DCs 48 2.3.5 In vitro activation and culture of T lymphocytes 50 2.3.6 [3H] thymidine proliferation assay 50 2.3.7 Annexin V/ 7-AAD viability assay 51 2.3.8 Viability & cell count assay 51 2.3.9 CFSE staining of T lymphocytes 53 2.3.10 Multiplex bead array 53 2.3.11 T cell proliferation with anti-mouse IL-2 neutralizing antibody 55 2.3.12 Depletion of CD4+CD25+T cells from CD4+cells 55 2.3.13 OVA peptide proliferation assay 56 2.3.14 Purification of naïve and memory CD8 and CD4 T cells 57 2.3.15 In vivo BrdU incorporation 57 2.3.16 In vitro BrdU incorporation and staining 58 2.3.17 DNA content analysis with PI staining 59 ii  2.3.18 Bradford assay to determine protein concentration 59 2.3.19 Preparation of 12% SDS-PAGE Gel 60 2.3.20 SDS gel electrophoresis and transfer onto nitrocellulose membrane 61 2.3.21 Western blot 61 2.3.22 Intracellular staining of cells 63 2.3.23 Statistical analysis 64 Chapter - Setting up in vitro model to study T cell expansion 65 3.1 Introduction 65 3.2 Purity of MACS purified CD8 and CD4 T cells from spleen & lymph nodes 67 3.3 Purity of MACS purified splenic CD11c+ DCs 69 3.4 Expression of co-stimulatory molecules on freshly isolated CD11c+ DCs 71 3.5 Optimising activation conditions for CD8 and CD4 T cells 73 3.6 CD8 and CD4 cell concentration & viablity over 72hrs with anti-CD3ε and CD11c+ DCs 76 3.7 CFSE proliferation of CD8 and CD4 T cells after 48hrs activation 80 3.8 Discussion 82 Chapter – Factors affecting T cell proliferation 85 4.1 Introduction 85 4.1 CD3ε and CD28 expression on CD8 and CD4 T cells 86 4.2 Effects of anti-CD3e alone/ with anti-CD28 on T cell proliferation 89 4.3 IL-2 and T cell proliferation 91 4.4 Effects of blocking IL-2 on T cell expansion 94 4.5 Expression of IL-2 receptor subunits on CD8 and CD4 T cells 96 4.6 CD4+ CD25+ T cells depletion on CD4 proliferation 99 4.7 CD8 and CD4 T cell proliferation in OVA-specific system 101 4.8 CD8 and CD4 T cell proliferation in BALBC mice 104 4.9 Heterogeneity within CD8 and CD4 T cell population 106 4.10 Proliferation of cell-sorted naïve CD8 and CD4 T cells 108 4.11 Discussion 111 Chapter – Kinetics of CD8 and CD4 T cells proliferation 117 5.1 Introduction 117 5.2 Resting naïve CD8 and CD4 T cells are in the same stage of the cell cycle 121 5.3 Kinetics of CD8 and CD4 T cell proliferation 126 5.4 Discussion 138 iii  Chapter – The effects of p27kip1 on CD8 and CD4 proliferation 142 6.1 Introduction 142 6.2 Expression of p27 in resting naïve T cells 144 6.3 Comparison of phenotype between WT and p27KO mice 146 6.4 Comparison of WT and p27KO T cell proliferation kinetics 147 6.5 Comparison of proliferation between p27KO CD8 and CD4 T cells 154 6.6 Effects of p27 on distribution of CD8 and CD4 populations 156 6.7 Effects of p27 on CD28 expression 159 6.8 Discussion 161 Chapter – Final discussion 166 7.1 Discussion 166 7.2 Limitations of the study 177 7.3 Future Work 179 References 181  iv  List of Figures Figure 1.1 Simplified overview of the immune system Figure Concept of immunological protection and phases of a T cell response 18 Figure The four phases of the cell cycle 25 Figure 3.3 FACS profile of splenic CD11c+ DCs from C57BL6 mice after MACS positive selection 70 Figure 3.4 Expression of co-stimulatory molecules CD80 and CD86 on freshly isolated splenic CD11c+ DCs 72 Figure 3.5 [3H] thymidine incorporation of CD8 and CD4 T cells with varying concentrations of anti-CD28/ T cell: CD11c+ DC ratio after 48hrs 75 Figure 3.6.1 Cell concentrations (cells/µl) of CD8 and CD4 T cells over a 72hr activation period 77 Figure 3.6 Percentage of viable CD8 and CD4 cells over a 72hrs activation period 79 Figure 3.7.1 Proliferation of viable CFSE-stained CD8 and CD4 T cells over 48hrs 81 Figure 4.1.1 Expression of CD3ε and CD28 on CD8 and CD4 T cells isolated from pooled C57BL6 spleen and lymph nodes 88 Figure 4.2.1 3H thymidine incorporation of CD8 and CD4 T cells activated with anti-CD3ε alone or with anti-CD28 over a period of 120 hours 90 Figure 4.3.1 IL-2, IFN-γ and IL-4 in supernatant of activated T cells 48hrs after activation 93 Figure 4.4.1 Effect of blocking IL-2 on 3H thymidine incorporation of CD8 and CD4 T cells activated with anti-CD3ε and anti-CD28 over a period of 72hrs 95 Figure 4.5.1 Expression of IL-2 receptor subunits on CD8 and CD4 T cell activated with 1µg/ml anti-CD3ε and 0.5μg/ml anti-CD28 over 48hr period 98 Figure 4.6.1 Comparison of proliferation of CFSE-stained CD4+ T cells and CD4+ CD25- T cells 48hrs after activation 100 Figure 4.7.1 3H thymidine incorporation of OTI CD8 and OTII CD4 T cells over a period of 120hrs 102 v  Figure 4.7.2 Comparison of proliferation of CFSE-stained OT-I CD8 cells and OT-II CD4 T cells 48hrs after activation 103 Figure 4.8.1 Comparison of proliferation of CFSE-stained BALBC CD8 and CD4 T cells activated with 1µg/ml anti-CD3ε and CD11c+ DCs over 72hrs 105 Figure 4.9.1 Heterogeneity within CD8 and CD4 T cells population within the lymph nodes and spleens of naïve C57BL6 mice Cells were gated using FSC vs SSC plot followed by staining positive for CD3e 107 Figure 5.1.1 Simplified schematic of T cell signalling pathway showing the action of PMA and ionomycin in initiating T cells activation 120 Figure 5.2.1 Stage of the cell cycle of naïve CD8 and CD4 T cells in the naïve mouse 122 Figure 5.2.2 Brdu incorporation in naive CD4 and CD8 T cells from lymph nodes 24hrs after BrdU injection 124 Figure 5.2.3 Brdu incorporation in naive CD4 and CD8 T cells from spleen 24hrs after BrdU injection 125 Figure 5.3.1A CFSE proliferation of naïve CD8 and CD4 T cells activated with anti-CD3ε and CD11c+ DCs from 27-36hrs 127 Figure 5.3.1 B CFSE proliferation of naïve CD8 and CD4 T cells activated with anti-CD3ε and CD11c+ DCs from 39-48hrs 128 132Figure 5.3.3 BrdU incorporation of naïve CD8 and CD4 T cells activated with anti-CD3ε and CD11c DCs between 0-48hrs 133 Figure 5.3.4 Graphical representation of percentage of cells with BrdU incorporation for naïve CD8 and CD4 T cells activated with anti-CD3e and CD11c DCs between 0-48hrs 134 Figure 5.3.5 Comparison of CD8 proliferation with/ without co-culture with CD4 T cells 136 Figure 5.3.6 Comparison of CD4 proliferation with/ without co-culture with CD8 T cells 137 Figure 6.2 Expression of p27kip1 in resting naïve CD4 and CD8 T cells 145 Figure 6.3 1Phenotype of WT and p27KO mice 146 Table 6.3 Recorded weight & length of spleens of WT and their respective p27KO littermates 147 vi  Figure 6.4.1 CFSE proliferation of WT and p27KO naive T cells activated with anti-CD3ε and CD11c+ DCs from 30 -36hrs 149 Figure 6.4.2 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