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Characterization of processing bodies in T and B lymphocytes Tebaykina, Zinaida 2012

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CHARACTERIZATION OF PROCESSING BODIES IN T AND B LYMPHOCYTES  by  ZINAIDA TEBAYKINA  B.Sc. The University of British Columbia, 2008  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES (Microbiology & Immunology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  April 2012  © Zinaida Tebaykina, 2012  ABSTRACT Processing bodies (P-bodies) are cytoplasmic aggregates that contain translationally-repressed mRNAs in complex with repressor proteins (GW182, RCK/p54, and DCP1a), facilitate mRNA storage or degradation, and can be identified by the αGWbody (GWB) serum that detects several P-body proteins. The partitioning of mRNAs between a translationally-competent cytoplasmic pool and a translationally-repressed Pbody pool could be an important mechanism for dynamically controlling the synthesis of key proteins. Memory CD8+ T lymphocytes contain translationally-repressed RANTES and IFN-γ mRNAs, enabling the secretion of these cytokines within 30 minutes of T cell receptor (TCR) engagement. Although P-bodies have not been characterized in lymphocytes, I hypothesized that storage of RANTES and IFN-γ mRNAs in P-bodies could contribute to the ability of memory CD8+ T cells to mount rapid recall responses. Using immunoblotting, flow cytometry, and confocal microscopy, I established that T and B lymphocytes contain GWBs and express GW182, RCK/p54, and DCP1a, which are concentrated in cytoplasmic granules. Co-localization analysis identified multiple subsets of P-bodies, raising the possibility that P-bodies with different protein compositions have distinct functional properties. Moreover, I found that P-bodies partially dissociate and move towards the model immune synapse in both T and B lymphocytes. To explore the role of P-bodies in the recall response, I utilized the OT-I model to generate effector and memory CD8+ T lymphocytes in vitro. Compared to naïve CD8+ T cells from OT-I mice, effector T cells had elevated levels of P-body proteins and a greater number of P-bodies. In contrast, memory T cells had similar numbers of Pbodies as naïve T cells, but contained larger GWBs and RCK/p54 granules. Remarkably, ii  RANTES mRNA did not co-localize with P-bodies in memory T cells, but was distributed diffusely in the cytoplasm. Conversely, IFN-γ mRNA co-localized with GWBs and RCK/p54 granules in memory T cells. The abundance of P-body-targeting AU-rich elements (AREs) in IFN-γ mRNA and the absence of AREs in RANTES mRNA suggests that IFN-γ mRNA transcribed following activation of naïve T cells, is directed for storage into GWB+ RCK/p54+ P-bodies to be reused during the recall response, whereas RANTES mRNA is stored by an undefined P-body-independent mechanism.  iii  PREFACE I.  Manuscript in preparation  •  Title: Dual analysis of lymphocytes by flow cytometry and confocal microscopy  •  Authors: Zinaida Tebaykina, Michael R. Gold  •  Contributions of authors and collaborators: Zinaida Tebaykina developed the protocol and wrote the manuscript in consultation with Michael R. Gold (M.R.G.). M.R.G. also provided reagents and funding supported by his grant from the Canadian Institutes of Health Research.  •  II. •  Thesis chapters: 2-3, 6.  Manuscript in preparation Title: IFN-γ mRNA is stored in processing bodies of memory CD8+ T lymphocytes.  •  Authors: Zinaida Tebaykina, Kate Choi, Lisa C. Osborne, Ninan Abraham, and Michael R. Gold  •  Contributions of authors and collaborators: Zinaida Tebaykina proposed, carried out, analyzed, and wrote up the study in consultation with Michael R. Gold (M.R.G). Kate Choi performed immunoblotting analysis of P-body markers (Figures 4.2 and 4.8). Lisa C. Osborne (L.C.O.) implemented preliminary in vivo experiments using the OT-I model of immune memory. Ninan Abraham (N.A.) and L.C.O. provided valuable feedback and ideas on experimental design and data interpretation relating to memory CD8+ T lymphocytes. M.R.G. and N.A. provided reagents for the study, with the exception of the reference human αGWB iv  serum 18033, which was received via collaboration with Marvin Fritzler from the University of Calgary. M.R.G. Funding was provided by a grant from the Canadian Institutes of Health Research to M.R.G. •  III.  Thesis chapters: 2, 4-6.  UBC Research Ethics Board certificates  •  Rodent Biology and Husbandry certification number: RBH-431-08  •  Canadian Council on Animal Care (CCAC) / National Institutional Animal User Training (NIAUT) Program certification number: 1431  •  UBC Animal Care Committee approval certificate number: A07-0194  v  TABLE OF CONTENTS Abstract.............................................................................................................................. ii	
   Preface ............................................................................................................................... iv	
   Table of contents .............................................................................................................. vi	
   List of tables.................................................................................................................... xiii	
   List of figures .................................................................................................................. xiv	
   List of illustrations ........................................................................................................ xvii	
   List of abbreviations .................................................................................................... xviii	
   Acknowledgments ......................................................................................................... xxii	
   Dedication ..................................................................................................................... xxiv	
   Chapter 1: Introduction ................................................................................................... 1	
   1.1. Overview .................................................................................................................. 1	
   1.2. Phases of the adaptive immune response ................................................................. 2	
   1.3. Immunity mediated by memory CD8+ T lymphocytes ............................................ 4	
   1.3.1. Subsets of memory CD8+ T lymphocytes .......................................................... 4	
   1.3.2. Development of memory CD8+ T lymphocytes ................................................. 5	
   1.3.3. Properties of memory CD8+ T lymphocytes vs. naïve and effector cells ........ 10	
   1.4. Memory CD8+ T lymphocytes contain translationally repressed IFN-γ and RANTES mRNAs that are rapidly translated during the recall phase .......................... 12	
   1.4.1. RANTES .......................................................................................................... 12	
   1.4.2. IFN-γ ............................................................................................................... 14	
   1.5. Kinetics of mRNA expression ............................................................................... 16	
   1.6. MiRNAs and AREs can direct target mRNAs to P-bodies for storage and can facilitate subsequent reactivation of translation ............................................................ 17	
   1.6.1 P-bodies ........................................................................................................... 17	
   vi  1.6.2 MiRNAs ............................................................................................................ 23	
   1.6.3 AREs................................................................................................................. 24	
   1.7. P-bodies move on microtubules and could be involved in localized translation ... 25	
   1.8. NK cells have adaptive immune features due to an unknown mechanism ............ 26	
   1.9. Significance............................................................................................................ 27	
   1.10. Hypotheses and goals........................................................................................... 27	
   Chapter 2: Materials and methods................................................................................ 30	
   2.1. Cell culture ............................................................................................................. 30	
   2.2. Mice ....................................................................................................................... 31	
   2.3. Isolation of primary T and B lymphocytes ............................................................ 31	
   2.4. Immunoblot analysis of P-body protein expression............................................... 32	
   2.5. Dual analysis of T and B lymphocytes by flow cytometry and confocal microscopy .................................................................................................................... 33	
   2.5.1. Initial protocol development ........................................................................... 33	
   2.5.2. Protocol adaptation for mRNA fluorescent in situ hybridization (FISH) ....... 35	
   2.5.3. Analysis on P-body markers in T and B lymphocytes ..................................... 36	
   2.5.4. Co-localization analysis on P-body markers in T and B lymphocytes ........... 38	
   2.6. Analysis of P-bodies in primary B lymphocytes following activation .................. 38	
   2.6.1. Activation of primary B lymphocytes with αIgM and/or αCD40 and IL-4 ..... 38	
   2.6.2. Analysis of activation markers on B lymphocytes treated with αIgM and/or αCD40 and IL-4 ........................................................................................................ 39	
   2.6.3. Analysis of P-body markers in activated B lymphocytes ................................ 40	
   2.7. Differentiation of naïve OT-I CD8+ T lymphocytes into effector and memory CD8+ T lymphocytes..................................................................................................... 40	
   2.7.1 Differentiating bone marrow-derived dendritic cells ...................................... 40	
   2.7.2. Maturing BMDCs and loading with the OVA257-264 peptide SIINFEKL ......... 41	
    vii  2.7.3. Isolating naïve CD8+ T lymphocytes from OT-I mice .................................... 41	
   2.7.4. Activating naïve OT-I CD8+ T lymphocytes with SIINFEKL-loaded mature BMDCs...................................................................................................................... 42	
   2.7.5. Differentiating activated OT-I CD8+ T lymphocytes into effector CD8+ T lymphocytes ............................................................................................................... 42	
   2.7.6. Differentiating activated OT-I CD8+ T lymphocytes into memory CD8+ T lymphocytes ............................................................................................................... 43	
   2.8. Phenotypic and functional analysis of naïve, activated, effector, and memory OT-I CD8+ T lymphocytes..................................................................................................... 44	
   2.8.1. Analysis of surface markers on naïve, activated, effector, and memory OT-I CD8+ T lymphocytes by flow cytometry .................................................................... 44	
   2.8.2. Analysis of proliferation potential of naïve OT-I CD8+ T lymphocytes activated with mature SIINFEKL-loaded BMDCs by CFSE dilution assay ............. 45	
   2.8.3. Analysis of cytotoxic potential in effector and memory OT-I CD8+ T lymphocytes activated with SIINFEKL-loaded EL4 target cells by cytotoxicity assay ................................................................................................................................... 46	
   2.8.4. Analysis of proliferation potential in effector and memory OT-I CD8+ T lymphocytes activated with SIINFEKL-loaded EL4 target cells by CFSE dilution assay.......................................................................................................................... 47	
   2.8.5. Analysis of survival potential in effector and memory OT-I CD8+ T lymphocytes activated with SIINFEKL-loaded EL4 target cells .............................. 48	
   2.9. Analysis of P-body markers and mRNA localization to P-bodies in naïve, activated, effector, and memory OT-I CD8+ T lymphocytes........................................ 49	
   2.9.1. Dual analysis of P-body markers in naïve, activated, effector, and memory OT-I CD8+ T lymphocytes by flow cytometry and confocal microscopy .................. 49	
   2.9.2. Poly(A) mRNA FISH and dual analysis of naïve, activated, effector, and memory OT-I CD8+ T lymphocytes by flow cytometry and confocal microscopy .... 49	
   2.9.3. RANTES mRNA FISH and dual analysis of naïve, activated, effector, and memory OT-I CD8+ T lymphocytes by flow cytometry and confocal microscopy .... 51	
   2.9.4. FISH for dual analysis of IFN-γ mRNA in naïve, activated, effector, and memory OT-I CD8+ T lymphocytes by flow cytometry and confocal microscopy .... 52	
    viii  2.9.5 Analysis of RANTES and IFN-γ mRNA localization to P-bodies in naïve, activated, effector, and memory OT-I CD8+ T lymphocytes..................................... 52	
   2.10. Analysis of P-body localization in naïve OT-I CD8+ T lymphocytes during immune synapse formation on mature SIINFEKL-loaded BMDCs ............................. 53	
   2.11. Analysis of P-body localization in naïve OT-I CD8+ T lymphocytes during the first asymmetric division after contacting mature SIINFEKL-loaded BMDCs ........... 54	
   2.12. Analysis of P-body localization in effector and memory OT-I CD8+ T lymphocytes during immune synapse formation on mature SIINFEKL-loaded EL4 target cells ..................................................................................................................... 56	
   2.13. Statistics ............................................................................................................... 58	
   Chapter 3: Improved methodology for staining lymphocytes in suspension ............ 59	
   3.1. Introduction ............................................................................................................ 59	
   3.2. Reagents, solutions, and equipment ....................................................................... 61	
   3.2.1. Specific reagents and equipment .................................................................... 61	
   3.2.2. Solution recipes............................................................................................... 62	
   3.3. Protocol .................................................................................................................. 63	
   3.3.1. Day 1: Passage cells and make solutions ....................................................... 64	
   3.3.2. Day 2: Fix cells and stain with 1º Abs ............................................................ 64	
   3.3.3. Day 3: Stain cells with 2° Abs, mount onto slides, and analyze by flow cytometry ................................................................................................................... 67	
   3.3.4. Day 4: Seal coverslips and image by confocal microscopy ............................ 69	
   3.4. Representative Results ........................................................................................... 69	
   3.5. Discussion .............................................................................................................. 71	
   3.5.1. Critical steps and possible modifications ....................................................... 71	
   3.5.2. Conclusions ..................................................................................................... 73	
   Chapter 4: Analysis of P-bodies in T and B lymphocytes ........................................... 75	
   4.1. Introduction ............................................................................................................ 75	
    ix  4.2. Characterization of P-bodies in T lymphocytes ..................................................... 76	
   4.2.1. T lymphocytes contain GWBs and express the P-body markers GW182, RCK/p54, and DCP1a............................................................................................... 76	
   4.2.2. GWBs, GW182, RCK/p54, and DCP1a are localized to discrete cytoplasmic foci in CD4+ T lymphocytes ...................................................................................... 80	
   4.2.3. CD4+ T lymphocytes contain different P-body subsets .................................. 80	
   4.2.4. GWBs, GW182, RCK/p54, and DCP1a are localized to discrete cytoplasmic foci in CD8+ T lymphocytes ...................................................................................... 87	
   4.2.5. CD8+ T lymphocytes contain different P-body subsets .................................. 91	
   4.3. Characterization of P-bodies in B lymphocytes..................................................... 94	
   4.3.1. B lymphocytes contain GWBs and express P-body markers GW182, RCK/p54, and DCP1a................................................................................................................ 94	
   4.3.2. GWBs, GW182, RCK/p54, and DCP1a are localized to discrete cytoplasmic foci in B lymphocytes ................................................................................................ 97	
   4.3.2. B lymphocytes contain two subsets of P-bodies .............................................. 97	
   4.4. Differences in P-body subsets between T and B lymphocytes ............................ 103	
   4.5. Analysis of P-bodies in B lymphocytes following activation.............................. 106	
   4.6. Discussion ............................................................................................................ 112	
   4.6.1. Analysis of P-bodies in T and B lymphocytes ............................................... 112	
   4.6.2. Evaluation of P-body localization to the model immune synapse in B lymphocytes ............................................................................................................. 115	
   4.6.3. Analysis of P-bodies in activated B lymphocytes .......................................... 117	
   Chapter 5: The role of P-bodies in memory CD8+ T lymphocytes and their recall response.......................................................................................................................... 119	
   5.1. Introduction .......................................................................................................... 119	
   5.2. Phenotypic and functional characterization of naïve, activated, effector, and memory OT-I CD8+ T lymphocytes ........................................................................... 122	
    x  5.2.1. Naïve, activated, effector, and memory OT-I CD8+ T lymphocytes express correct surface markers .......................................................................................... 122	
   5.2.2. Naïve, activated, effector, and memory OT-I CD8+ T lymphocytes exhibit correct functional properties .................................................................................. 127	
   5.3. Comparison of P-bodies in naïve, activated, effector, and memory OT-I CD8+ T lymphocytes ................................................................................................................ 138	
   5.4. Analysis of mRNA localization to P-bodies in naïve, activated, effector, and memory OT-I CD8+ T lymphocytes ........................................................................... 141	
   5.4.1. Poly(A) mRNA is located in the nucleus and diffusely in the cytoplasm of naïve, activated, effector, and memory OT-I CD8+ T lymphocytes ........................ 141	
   5.4.2. RANTES mRNA is highly expressed and diffusely distributed in the cytoplasm of effector and memory OT-I CD8+ T lymphocytes ................................................ 141	
   5.4.3. IFN-γ mRNA localizes to GWBs and RCK/p54 granules in activated, effector, and memory OT-I CD8+ T lymphocytes ................................................................. 144	
   5.5. Analysis of P-body localization to the immune synapse in naïve, effector, and memory OT-I CD8+ T lymphocytes ........................................................................... 151	
   5.6. Analysis of P-body distribution during the first asymmetric division of naïve OT-I CD8+ T lymphocytes on SIINFEKL-loaded mature BMDCs .................................... 154	
   5.7. Discussion ............................................................................................................ 159	
   5.7.1. Characterization of phenotype and functional properties of naïve, activated, effector, and memory OT-I CD8+ T lymphocytes ................................................... 159	
   5.7.2. Comparison of P-bodies in naïve, activated, effector, and memory OT-I CD8+ T lymphocytes.......................................................................................................... 160	
   5.7.3. Analysis of mRNA localization to P-bodies in naïve, activated, effector, and memory OT-I CD8+ T lymphocytes ........................................................................ 162	
   5.7.4. Evaluation of P-body localization to the immune synapse in T lymphocytes 164	
   5.7.5. Assessment of P-body distribution during the first asymmetric division of naïve CD8+ T lymphocytes ..................................................................................... 165	
   Chapter 6: Discussion ................................................................................................... 167	
   6.1. Introduction .......................................................................................................... 167	
    xi  6.2. Main conclusions ................................................................................................. 168	
   6.2.1. Development of the protocol for dual analysis of proteins and/or mRNAs in lymphocytes by flow cytometry and confocal microscopy ...................................... 168	
   6.2.2. Analysis of P-bodies in T and B lymphocytes ............................................... 170	
   6.2.3. Characterization of phenotype and functional properties of naïve, activated, effector, and memory OT-I CD8+ T lymphocytes ................................................... 171	
   6.2.4. Comparison of P-bodies in naïve, activated, effector, and memory OT-I CD8+ T lymphocytes.......................................................................................................... 173	
   6.2.5. Analysis of P-bodies in activated B lymphocytes .......................................... 174	
   6.2.6. Analysis of mRNA localization to P-bodies in naïve, activated, effector, and memory OT-I CD8+ T lymphocytes ........................................................................ 174	
   6.2.7. Evaluation of P-body localization to the immune synapse in T and B lymphocytes ............................................................................................................. 175	
   6.2.8. Assessment of P-body distribution during the first asymmetric division of naïve CD8+ T lymphocytes ..................................................................................... 177	
   6.3. Future directions .................................................................................................. 178	
   6.4. Significance.......................................................................................................... 182	
   Bibliography .................................................................................................................. 183	
    xii  LIST OF TABLES Table 2.1. Staining of immune synapses formed by naïve OT-I CD8+ T lymphocytes with SIINFEKL-loaded mature BMDCs for 20 minutes, 1 hour, and 4 hours ................. 54 Table 2.2. Staining the first asymmetric division of naïve OT-I CD8+ T lymphocytes on SIINFEKL-loaded mature BMDCs following 28 hours of co-culture ............................. 57 Table 3.1. List of specific reagents and equipment .......................................................... 61 Table 3.2. Recipe for FACS buffer .................................................................................. 62 Table 3.3. Recipe for Flow buffer .................................................................................... 62 Table 3.4. Recipe for saponin wash buffer (SWB) .......................................................... 62 Table 3.5. Recipe for antibody buffer (Ab buffer) ........................................................... 62 Table 3.6. Recipe for complete RPMI medium ............................................................... 62 Table 3.7. Sample calculation for counting cells before fixation..................................... 65 Table 3.8. Sample plate outline for staining cells in suspension...................................... 67 Table 3.9. Sample calculation for staining cells in suspension with primary antibodies . 67 Table 3.10. Sample calculation for staining cells in suspension with secondary antibodies and rhodamine-phalloidin ................................................................................................. 68  xiii  LIST OF FIGURES Figure 1.1. Memory CD8+ T lymphocytes respond faster during the recall phase than during the initial activation phase ....................................................................................... 3 Figure 1.2. Models for the development of a single naïve CD8+ T lymphocyte into effector and memory cells ................................................................................................... 8 Figure 1.3. Phenotypic and functional properties of naïve, effector, and memory CD8+ T lymphocyte........................................................................................................................ 13 Figure 1.4. Pre-existing RANTES mRNA in memory CD8+ T lymphocytes is translated during the recall response ................................................................................................. 11 Figure 1.5. Kinetics of mRNA expression during an inflammatory response and during the development of memory CD8+ T lymphocytes .......................................................... 18 Figure 1.6. MiRNAs are able to direct target mRNAs into P-bodies for storage and subsequent recruitment for translation ............................................................................. 19 Figure 1.7. P-bodies share some of their protein components with other RNA granules 22 Figure 3.1. Dual analysis of lymphocytes by flow cytometry and confocal microscopy allows to visualize fine intracellular details, document consistency of staining, and determine protein expression levels .................................................................................. 70 Figure 3.2. Dual analysis of lymphocytes by flow cytometry and confocal microscopy can be adapted for FISH ................................................................................................... 74 Figure 4.1. Primary CD4+ and CD8+ T lymphocytes were isolated to high purity from WT mice and contain a subset of memory-phenotype cells ............................................. 78 Figure 4.2. CD4+ and CD8+ T lymphocytes express GW182, RCK/p54, DCP1a, and the P-body markers detected by the reference anti-GWB serum............................................ 79 Figure 4.3. Primary murine CD4+ T lymphocytes contain GWBs and express GW182, RCK/p54, and DCP1a that are localized to discrete cytoplasmic foci ............................. 82 Figure 4.4. Primary murine CD4+ T lymphocytes contain subsets of P-bodies with various compositions of GWBs, GW182, RCK/p54, and DCP1a ................................... 85 Figure 4.5. Primary murine CD8+ T lymphocytes contain GWBs and express GW182, RCK/p54, and DCP1a that are localized to discrete cytoplasmic foci ............................. 89  xiv  Figure 4.6. Primary murine CD8+ T lymphocytes contain subsets of P-bodies with various compositions of GWBs, GW182, RCK/p54, and DCP1a ................................... 92 Figure 4.7. Primary B lymphocytes were isolated to high purity from WT mice ........... 95 Figure 4.8. B lymphocytes express GW182, RCK/p54, DCP1a, and the P-body markers detected by the reference anti-GWB serum ...................................................................... 96 Figure 4.9. Primary murine B lymphocytes contain GWBs and express GW182, RCK/p54, and DCP1a that are localized to discrete cytoplasmic foci ............................. 99 Figure 4.10. Primary murine B lymphocytes contain two subsets of P-bodies: GWB+ RCK/p54+ DCP1a+ and GW182+ .................................................................................... 101 Figure 4.11. Differences in P-body subsets between T and B lymphocytes ................. 104 Figure 4.12. Changes in P-bodies of primary murine B lymphocytes following activation with αIgM and/or αCD40 and IL-4 ................................................................................. 110 Figure 5.1. Naïve, activated, effector, and memory OT-I CD8+ T lymphocytes display correct phenotype ........................................................................................................... 124 Figure 5.2. In contrast to naïve cells, effector and memory OT-I CD8+ T lymphocytes display cytotoxicity towards EL4 target cells that present SIINFEKL on H-2Kb .......... 130 Figure 5.3. In contrast to effector cells that were previously cultured with IL-2, naïve and memory OT-I CD8+ T lymphocytes do not proliferate in 24 hours following on TCR engagement and/or stimulation with IL-2 ...................................................................... 132 Figure 5.4. In contrast to naïve cells, effector and memory OT-I CD8+ T lymphocytes can be induced to proliferate in 48 hours via stimulation with IL-2 .............................. 134 Figure 5.5. In contrast to naïve and effector cells, memory OT-I CD8+ T lymphocytes are able to survive up to 48 hours without TCR engagement and stimulation with IL-2 .... 137 Figure 5.6. Memory OT-I CD8+ T lymphocytes contain large GWBs and RCK/p54 granules that are absent in naïve cells, but contain similar numbers of P-bodies ........... 140 Figure 5.7. In naïve, activated, effector, and memory OT-I CD8+ T lymphocytes, poly(A) mRNA is located in the nucleus and distributed diffusely in the cytoplasm, but not in cytoplasmic granules ....................................................................................................... 142 Figure 5.8. RANTES mRNA is highly expressed and diffusely distributed in the cytoplasm of effector and memory OT-I CD8+ T lymphocytes ..................................... 146  xv  Figure 5.9. IFN-γ mRNA is expressed and localized to cytoplasmic granules in activated, effector, and memory OT-I CD8+ T lymphocytes .......................................................... 148 Figure 5.10. IFN-γ mRNA co-localize with GWBs in activated, effector, and memory OT-I CD8+ T lymphocytes.............................................................................................. 150 Figure 5.11. GWBs localize in proximity of the immune synapse and GW182 is located at the immune synapse formed by naïve OT-I CD8+ T lymphocytes with SIINFEKLloaded mature BMDCs over 1 hour ................................................................................ 152 Figure 5.12. GWBs localize in proximity of the immune synapse formed by effector or memory OT-I CD8+ T lymphocytes with SIINFEKL-loaded EL4 target cells over 1 hour ......................................................................................................................................... 153 Figure 5.13. Naïve OT-I CD8+ T lymphocytes start dividing between 28 and 34 hours following activation by SIINFEKL-loaded mature BMDCs .......................................... 155 Figure 5.14. Localization of GWBs and GW182 right before and during the first asymmetric division of naïve OT-I CD8+ T lymphocytes on SIINFEKL-loaded mature BMDCs ........................................................................................................................... 158  xvi  LIST OF ILLUSTRATIONS Illustration 1.1. Overall hypothesis ................................................................................. 29 Illustration 3.1. Method outline for dual analysis of calnexin and α-tubulin in murine CD8+ T-cell line CTLL2 by flow cytometry and confocal microscopy ........................... 63 Illustration 4.1. Method outline for dual analysis of P-body markers in primary murine CD4+ T lymphocytes by flow cytometry and confocal microscopy ................................. 81 Illustration 4.2. Method outline for dual analysis of P-body markers in primary murine CD8+ T lymphocytes by flow cytometry and confocal microscopy ................................. 88 Illustration 4.3. Method outline for dual analysis of P-body markers in primary murine B lymphocytes by flow cytometry and confocal microscopy .............................................. 98 Illustration 4.4. Method outline for analysis of P-bodies in primary murine B lymphocytes activated with αIgM and/or αCD40 and IL-4 ........................................... 107 Illustration 5.1. Method outline for generating activated, effector, and memory CD8+ T cells from naïve OT-I CD8+ T lymphocytes in vitro ...................................................... 121 Illustration 5.2. Method outline for cytotoxicity assay with effector or memory OT-I CD8+ T lymphocytes and CellTracker Orange-labeled SIINFEKL-loaded EL4 target cells ......................................................................................................................................... 128 Illustration 5.3. Method outline for a combined assay to analyze long-term cytotoxicity, proliferation, and survival of effector or memory OT-I CD8+ T lymphocytes via coculture with CellTracker Orange-labeled SIINFEKL-loaded EL4 target cells .............. 129 Illustration 6.1. Method outline for dual analysis of proteins and/or mRNAs by flow cytometry and confocal microscopy ............................................................................... 169 Illustration 6.2. P-body subsets in primary T and B lymphocytes ............................... 172 Illustration 6.3. Proposed model for the storage of effector cytokine mRNAs in memory CD8+ T lymphocytes....................................................................................................... 176  xvii  LIST OF ABBREVIATIONS American Type Culture Collection  ATCC  Antibody  Ab  Antigen presenting cell  APC  Argonaute 2  AGO2  Atypical protein kinase C  aPKC  AU-rich element-binding factor 1  AUF1  AU-rich element-binding proteins  ARE-BPs  AU-rich elements  AREs  B cell receptor  BCR  Bicinchoninic acid  BCA  Bone marrow-derived dendritic cell  BMDC  Bovine serum albumin, fraction V  BSA  Butyrate response factor 1  BRF1  C57BL/6  B6  C57BL/6-Tg(TcraTcrb)1100Mjb/J  OT-I  Chemokine C-C motif ligand 5  CCL5  Carboxyfluorescein diacetate succinimidyl ester  CFDA-SE  Carboxyfluorescein succinimidyl ester  CFSE  Cationic amino acid transporter 1  CAT-1  Central memory T cell  TCM  Central supramolecular activation complex  cSMAC  Cross-linking and immunoprecipitation  CLIP  Cytotoxic T lymphocyte  CTL  xviii  Dendritic cell  DC  4',6-diamidino-2-phenylindole  DAPI  Digoxigenin  DIG  Dilution factor  DF  Dulbecco's modified Eagle medium  DMEM  Effector memory T cell  TEM  Endoplasmic reticulum  ER  Enhanced chemiluminescence  ECL  Extracellular matrix  ECM  Fetal bovine serum  FBS  Fluorescein isothiocyanate  FITC  Fluorescent in situ hybridization  FISH  Follicular helper T cell  TFH  Forward scatter  FSC  Fragile X mental retardation-related protein 1  FXR1  Granulocyte-macrophage colony-stimulating factor  GM-CSF  Horseradish peroxidase  HRP  Hu protein R  HuR  Human embryonic kidney 293  HEK 293  Hypoxia inducible factor 1α  HIF-1α  Immunoglobulin  Ig  Inducible costimulator  ICOS  Interferon  IFN  Intracellular adhesion molecule  ICAM  Interleukin  IL  xix  KH-type splicing regulatory protein  KHSRP  Lipopolysaccharide  LPS  Locked nucleic acid  LNA  Lymphocyte function-associated antigen-1  LFA-1  Major histocompatibility complex  MHC  Mean fluorescent intensity  MFI  Memory precursor effector cell  MPEC  Memory-phenotype  MP  MicroRNA  miRNA  MicroRNA-loaded RNA-induced silencing complex  miRISC  Microtubule organizing center  MTOC  Microtubules  MTs  Mitogen-activated protein kinase  MAPK  Monoclonal antibody  mAb  Multivesicular body  MVB  Natural killer  NK  Ovalbumin  OVA  Pacific blue  PB  Paraformaldehyde  PFA  Pattern recognition receptor  PRR  Pathogen-associated molecular pattern  PAMPs  Peripheral supramolecular activation complex  pSMAC  Phorbol 12-myristate 13-acetate  PMA  Phosphate-buffered saline, pH 7.4  PBS  Phycoerythrin  PE  xx  Polymerase chain reaction  PCR  Protein kinase C  PKC  Regulated upon activation, normal T-cell expressed, and secreted  RANTES  Regulatory T cell  TReg  RNA-induced silencing complex  RISC  Room temperature  RT  Saponin FISH buffer  SFB  Saponin wash buffer  SWB  Short-lived effector cell  SLEC  Side scatter  SSC  Saline sodium citrate  SSC  Signal transducer and activator of transcription  STAT  Sodium dodecyl sulfate polyacrylamide gel electrophoresis  SDS-PAGE  Standard error of the mean  SEM  Standard deviation  SD  Stem cell memory T cell  TSCM  T cell receptor  TCR  Tissue culture  TC  Toll-like receptor  TLR  Tris buffered saline, pH 7.4  TBS  Tris buffered saline, pH 7.4 containing 0.1% Tween 20  TBST  Tristetraprolin  TTP  Tumor necrosis factor  TNF  3′ untranslated region  3’UTR  xxi  ACKNOWLEDGMENTS I really appreciate the enthusiasm, guidance, and friendship of every person who made my graduate school experiences such a pleasant journey. My biggest gratitude goes to Dr. Michael R. Gold, who took the time to listen to my ideas, was a patient and caring supervisor, and encouraged me to work on the exciting P-body project, even though it was a new direction for the lab. I would also like to thank my committee members Dr. Ninan Abraham and Dr. Eric Jan for their scientific insight, ideas, and making the time to meet with me whenever I needed their expertise. Without them, this thesis might have been very different. I thank Dr. Hung-Sia Teh and Dr. Ken Harder for encouraging me to show my MICB 502 grant proposal about the role of P-bodies in immune memory to Dr. Gold and a suggestion to pursue the idea in the lab. In addition, I appreciate Dr. Harder’s feedback on one of the protocols and his attendance at my thesis defense as an external examiner. Of course, I thank my past and present labmates: Kathrin Brenker, Kate Choi, Sonja Christian, May Dang-Lawson, Dr. Raibatak Das, Dr. Caylib Durand, Spencer Freeman, Megan Gilmour, Crystal Lee, Victor Lei, Dr. Kevin Lin, Dr. Marcia McCoy, Dr. Kathy Tse, and Jia Wang, who made my graduate school years such a unique experience. I especially thank Kate, May, Spencer, and Kevin (Gold lab), Dr. Lisa Osborne (Abraham lab), Anthony Khong (Jan lab), Dr. Amy Saunders and Dr. Nina Maeshima (Johnson lab), Munreet Chehal (Harder lab), and Dr. Edwin Moore for teaching me experimental techniques, helping with experiments, and/or great scientific  xxii  discussions. It was also a pleasure to share the lab with our work-study students Alex, Denny, and Helen, as well as with our directed studies students Aileen and Melanie. I am grateful to Dr. Marvin Fritzler and Meifeng Zhang from the University of Calgary for providing the reference human αGWB serum 18033, a crucial reagent for studying P-bodies. I also thank Justin Wong and Andy Johnson of the UBC Flow Cytometry Facility, as well as Goran Mansson, Pascal St-Pierre, Min Fu, and Dr. Robert Nabi of the Life Sciences Institute Imaging facility for training and technical assistance. Funding for this work was provided by a grant from the Canadian Institutes of Health Research to Dr. Gold and by my Graduate Entrance Scholarship and International Partial Tuition Scholarships from UBC. My immense gratitude goes to my parents and brothers: Nina, Alex, Gleb, and Dmitry Tebaykins, as well as to my boyfriend Otavio Good, for their love that made my life as incredible as it is. Finally, to follow Victor Lei’s tradition, I thank you, kind reader, for taking the time to look at my thesis.  xxiii  DEDICATION This thesis is dedicated to Otavio Good and the Tebaykins family.  xxiv  CHAPTER 1: INTRODUCTION 1.1. Overview The mammalian immune response to infection is mediated by the innate and the adaptive immune systems (reviewed in Refs. 1-4). Cells of the adaptive immune system possess two important characteristics: antigen specificity and an amplified response upon subsequent exposure, which together form the basis for immunological memory. Translationally-repressed messenger RNAs (mRNAs) that encode two key effector proteins of the T-cell immune response, RANTES and interferon (IFN)-γ, were identified inside resting memory CD8+ T lymphocytes 5-7, as well as inside the recently characterized memory-like natural killer (NK) cells 8-10, suggesting a role for these stored effector mRNAs in immunological memory. This led me to hypothesize that RANTES and IFN-γ mRNAs are stored in mRNA processing bodies (P-bodies), which are cellular structures specialized in mRNA storage and turnover (reviewed in Refs. 11-14). P-bodies have been studied in several cell types, but have not yet been characterized in lymphocytes. The aim of this project was to determine whether P-bodies exist in T and B lymphocytes and to assess the role of P-bodies in immunological memory by investigating whether RANTES and IFN-γ mRNAs were stored within P-bodies in memory CD8+ T cells. Understanding of immunological memory could aid the development of effective vaccines, and this study investigates a possible mechanism by which memory CD8+ T lymphocytes are able to respond to secondary infections faster than naïve cells.  1  1.2. Phases of the adaptive immune response Activation of a rare, antigen (Ag)-specific lymphocyte of the adaptive immune system, such as a T cell, occurs shortly after primary infection (reviewed in Refs. 1-4) (Figure 1.1). This activation phase is followed by rapid proliferation of Ag-specific lymphocytes into primary effector cell populations, which is known as the expansion phase. Most (90–95%) of these primary effector cells undergo apoptosis during the contraction phase, after the infection is cleared. During the memory phase, the surviving lymphocytes form a pool of long-lived populations that patrol lymphoid and non-lymphoid tissues. If a memory lymphocyte re-encounters its cognate Ag, the recall phase (also known as the secondary immune response, or the recall response) is initiated. During the recall phase, memory lymphocytes rapidly differentiate into effector cells, such that they secrete effector cytokines, mediate other effector functions (e.g. cytolysis), and proliferate more rapidly than during the initial activation phase 15-18. Several mechanisms that could explain these differences in kinetics of the activation and the recall phases have been proposed: accumulation of specific transcription factors 18, altered expression of surface receptors 17, increased signal strength due to affinity maturation and changes in the cytoplasmic domain of the B-cell Ag receptor (BCR) in B lymphocytes 4, epigenetic DNA modifications 19, 20, and changes in the subnuclear compartmentalization of genetic loci 21. However, it still remains to be determined why a cognate Ag induces the expression of effector proteins more rapidly in memory cells than in naïve cells.  2  Figure 1.1. Memory CD8+ T lymphocytes respond faster during the recall phase, than during the initial activation phase (adapted from Ref. 2). Activation phase (a), expansion phase (b), contraction phase (c), memory phase (d and e), and recall phase (f) of the adaptive immune response. CD8+ T lymphocytes are shown as an example. A rare, antigenspecific naïve CD8+ T lymphocyte is activated by a DC presenting a peptide from Ag on MHC I during the activation phase. In expansion phase, this naïve cell rapidly proliferates to give rise to effector CD8+ T lymphocytes and memory precursor cells. Most of these cells undergo apoptosis during the contraction phase, after the infection is cleared. During the memory phase, the surviving cells form a pool of long-lived memory CD8+ T lymphocytes that patrol lymphoid and non-lymphoid tissues. The recall phase is initiated if a memory CD8+ T lymphocyte re-encounters its cognate peptide-MHC I complex, enabling rapid clearance of infection.  3  1.3. Immunity mediated by memory CD8+ T lymphocytes Memory CD8+ T lymphocytes are self-renewing, Ag-experienced CD8+ T cells that maintain a capacity for clonal expansion in persistent or recurring infections (reviewed in Refs. 1-4, 22). Because of their ability to rapidly respond to invaders, such as bacteria, viruses, other microbial molecules, or even cancer cells, understanding the development and function of memory CD8+ T lymphocytes is a long-standing goal of immunology. 1.3.1. Subsets of memory CD8+ T lymphocytes Based on differential expression of the CCR7 and CD62L lymphoid homing receptors, Ag-experienced CD8+ T lymphocytes were originally divided into two subsets: central memory (TCM; CCR7+ and CD62LHigh) and effector memory (TEM; CCR7- and CD62LLow) 23. TEM cells are normally present in spleen, blood, and non-lymphoid tissues, but not in lymph nodes. TCM cells can be found in lymph nodes, spleen, and blood. In addition, TCM cells can home to non-lymphoid tissues, even though less efficiently than TEM cells 24-26. CD8+ TEM and TCM cells are nearly equivalent in their proliferation, production of effector cytokines, and cytotoxicity in response to T-cell receptor (TCR) ligation 23, 26-29. However, only TCM cells are able to proliferate in response to homeostatic signals, such as cytokines interleukin (IL)-7 and IL-15, and home to secondary lymphoid organs during re-infection for secondary clonal expansion 23, 30. Although still a subject of debate, TEM cells seem to mount a better recall response at early time points after viral infection, while TCM cells respond more vigorously to secondary challenge at later timepoints 31, 32. TEM cells can be further subdivided into transitional TEM cells, which are  4  relatively long-lived and can further differentiate into TCM cells, and end-stage TEM cells, which are relatively short-lived and patrol peripheral tissues to prevent immediate reinfection (reviewed in Ref. 33). More recently, a stem cell memory T-cell subset (TSCM; CD44Low CD62LHigh CD122High Sca-1+) has been described among Ag-experienced CD8+ T lymphocytes in mice 34, 35. TSCM cells are capable generating both TCM and TEM subsets, while selfrenewing themselves. Remarkably, adoptively transferred TSCM cells confer superior immunity compared to either TCM or TEM subsets 34. Remarkably, a novel subset on non-recirculating resident memory CD8+ T (TRM) lymphocytes was discovered in skin earlier this year (2012) 36, 37. These TRM cells populate both the previously infected and non-involved skin, do not enter the circulation, and provide a better protection than TCM cells during the recall phase 36, 37. Finally, memory-phenotype (MP) CD8+ T lymphocytes are a population of CD44High CD8+ T cells found in naïve animals (reviewed in Ref. 38). MP T cells are present in germ-free mice, and therefore it is unlikely that these cells are the result of interaction with normal flora 39. Instead, MP CD8+ T cells are thought to be generated via lymphopenia-induced proliferation when the first mature T cells exit the thymus in neonatal mice 40-42. MP CD8+ T lymphocytes mount a better recall response than naïve cells, but are less efficient than Ag-experienced cells in responding to secondary infection, and thus are not considered true memory cells 43. 1.3.2. Development of memory CD8+ T lymphocytes Early in infection, Ag-presenting cells (APCs), such as dendritic cells (DCs) and 5  macrophages, encounter Ags derived from invading microbes and present peptides from these Ags on major histocompatibility (MHC) I molecules to naïve CD8+ T lymphocytes. This occurs primarily in lymph nodes draining the site of infection, or in the spleen if the infection is blood-borne. If one of the ~100-200 Ag-specific naïve CD8+ T cells in the body recognizes the peptide-MHC I complex via its TCR, receives co-stimulatory signals from the APC (e.g. CD80/CD86), and encounters cytokines (e.g. IL-2) from a T helper (TH) cell, this Ag-specific naïve CD8+ T cell will undergo rapid proliferation in the secondary lymphoid organ to give rise to ~106-107 effector CD8+ T lymphocytes that migrate to the periphery to resolve the infection through cytotoxic activity (via perforin and granzymes) and the secretion of cytokines, such as IFN-γ and tumor necrosis factor (TNF)-α 30, 33, 44 (reviewed in Refs. 33, 45). At the peak of the primary immune response, Ag-specific effector CD8+ T lymphocytes can be subdivided into two main subpopulations: memory precursor effector cells (MPECs; CD127High KLRG1Low) and short-lived effector cells (SLECs; CD127Low KLRG1High) 46. SLECs are either shortlived, or relatively long-lived but senescent, and therefore are unable to maintain the much-needed clonal expansion if the infection is persistent (e.g. HCV or HIV), enters a latent phase (e.g. HHV), or is re-encountered later as a secondary infection. To offer protection in the above cases, naïve CD8+ T lymphocytes also give rise to long-lived memory cells (Figure 1.1; reviewed in Ref. 22). Upon reinfection, memory CD8+ T lymphocytes begin to rapidly produce effector molecules, proliferate, and differentiate into secondary effector cells. This qualitatively and quantitatively (the number of Agspecific memory CD8+ T lymphocytes is ~103-104 times higher than the number of naïve cells) enhanced recall response results in a better control of infection compared to the  6  primary response (reviewed in Ref. 33). How activation of a single naïve CD8+ T lymphocyte can lead to the development of both effector and memory cells remains an active area of research. This cell fate decision could be determined by extrinsic factors or TCR signal strength (Figure 1.2, AD; reviewed in Ref. 33). However, the finding that the first division of an Ag-specific naïve CD8+ T lymphocyte following activation by an Ag-bearing mature DC can be asymmetric, giving rise to molecularly distinct daughter cells with different effector and memory fate potential 47, the asymmetric cell division model received strong support 48, 49  . It was found that daughter cells that were proximal to the immunological synapse had  phenotypic characteristics of effector CD8+ T lymphocytes, while APC-distal daughter cells had phenotype of memory cells and were able to protect mice from infection 30 days later 47. However, because both daughter cells are of similar size immediately following the first division 47, 50, it is more probable that their identities are SLEC and MPEC, rather than mature effector and memory cells, respectively, because mature memory CD8+ T cells are considerably smaller than mature effector cells 51, 52. An observation that distal daughter cells contain large amounts of IL-7Rα (CD127) mRNA 47 confirms their identity as MPECs and supports the asymmetric cell division model (Figure 1.2, E). A subsequent report confirmed that the initial division of naïve CD8+ T cells is asymmetric and found that this first division takes place while the T cell is still attached to the APC 50. Overall, it has been shown that the proximal daughter cell (SLEC phenotype) is enriched in the Scribble 47, 50, DlgF 50, protein kinase C (PKC)-θ 50, and PKC-ζ 47 polarity proteins, whereas the distal daughter cell is enriched in a distinct set of polarity-associated proteins that includes atypical PKC (aPKC) 50, Par3 50, Numb 47, 50,  7  Figure 1.2. Models for the development of a single naïve CD8+ T lymphocyte into effector and memory cells (adapted from Refs. 33, 49). 8  (A) Uniform potential model. All activated CD8+ T cells become MPECs (blue), but the number of memory cells generated is limited by extrinsic factors. MPECs give rise to transitional TEM cells (gray) that later become self-renewing TCM cells (violet). (B) Decreasing potential model. Shorter TCR signal favors the development of TCM cells from MPECs, while longer TCR stimulation promotes terminal differentiation into SLECs (yellow) and TEM cells (green) that decline over time. (C) Fixed lineage model. Activation of a naïve CD8+ T cell results into the development of either selfrenewing TCM cells, or into SLECs. (D) Fate commitment with progressive differentiation model. Stronger TCR signal at the time of activation results into formation of SLECs, while weaker TCR signal induces the development of MPECs. Most SLECs die by apoptosis, but some persist with a limited lifespan as end-stage TEM cells. Even though MPECs acquire effector functions, they remain multipotent and give rise to more SLECs and to transitional TEM cells that progressively mature into self-renewing TCM cells. (E) Asymmetric division model. The fate of a naïve CD8+ T cell to become either SLEC or MPEC is determined at the time of the first division following activation due to asymmetric distribution of key proteins. The daughter cell proximal to the immune synapse becomes SLEC, while the distal daughter cell becomes MPEC. Note that this model is compatible with (D) following this first division.  9  CD45 50, and Pins 50. Because asymmetric distribution of IL-7Rα mRNA and possibly other mRNAs during the first division of naïve CD8+ T lymphocytes could be an important mechanism for MPEC vs. SLEC fate decision, this study aimed at determining the distribution of several mRNA interacting proteins during this first asymmetric division. 1.3.3. Properties of memory CD8+ T lymphocytes vs. naïve and effector cells A hallmark of memory CD8+ T lymphocytes is their ability to survive in an Agindependent manner and undergo homeostatic turnover (slow division) (Figure 1.3) 33, 53. Notably, exposure to IL-15 during the expansion phase and to IL-7 during the contraction phase is important for long-term survival of memory CD8+ T lymphocytes (reviewed in Ref. 38). Effector CD8+ T lymphocytes contain high levels of the cytotoxic granule molecules, perforin and granzyme B, whereas memory cells contain very little or none 7, 26  . Nevertheless, in contrast to naïve CD8+ T lymphocytes, memory cells are able to  respond with surprising speed during the recall phase, such that killing of target cells in vivo is evident starting at 1 hour after re-infection (the earliest timepoint examined) 28. It is important to note that memory CD8+ T lymphocytes are weakly cytotoxic in vitro, but are able to induce the death of target cells almost as well as effector cells in vivo 28. Moreover, naïve CD8+ T lymphocytes are unable to produce the effector cytokines IFN-γ or TNF-α for at least 5 hours following TCR stimulation, whereas effector and memory cells are able to produce these cytokines in ≤ 30 minutes and ≤ 4 hours in response to TCR engagement, respectively. 54, 55 Finally, resting naïve, effector, and memory CD8+ T lymphocytes do not synthesize the effector cytokines IFN-γ and TNF-α. 54, 55  10  Figure 1.3. Phenotypic and functional properties of naïve, effector, and memory CD8+ T lymphocytes (adapted from Ref. 30). Comparison of naïve, effector, and memory CD8+ T cells in homing to secondary (2º) lymphoid organs and peripheral tissues, cytotoxic T lymphocyte (CTL) ability, proliferation and cytokine secretion following TCR engagement, proliferation due to homeostatic cytokines IL-7 and IL-15, and markers. 30, 33, 55-59.  11  1.4. Memory CD8+ T lymphocytes contain translationally repressed IFN-γ and RANTES mRNAs that are rapidly translated during the recall phase 1.4.1. RANTES Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES), also known as Chemokine C-C motif Ligand 5 (CCL5), belongs to the family of C-C chemokines and is a potent proinflammatory chemoattractive cytokine (chemokine). It is expressed by CD8+ T lymphocytes at later times (5-7 days) following TCR-induced activation of naïve T cells 60. RANTES acts on multiple cell types that express the chemokine receptors CCR1 61, 62, CCR3 62, 63, CCR5 61, 62, 64, and GPR75 65. In contrast to their naïve counterparts, resting memory CD8+ T lymphocytes contain high levels of spliced cytoplasmic mRNA encoding RANTES 5, 6 (Figure 1.3A). These memory cells do not contain intracellular RANTES protein or secrete the RANTES until they are activated by TCR signaling 5. Secretion of RANTES by memory CD8+ T cells requires translation, but not transcription 5, 6, suggesting that TCR engagement induces the translation of pre-existing RANTES mRNA during this recall response (Figure 1.3B). The ability to start secreting RANTES within 30 minutes of TCR stimulation 6 allows memory CD8+ T lymphocytes to rapidly attract DCs and macrophages to the site of infection and enhance their maturation 66, 67. RANTES also acts on natural killer (NK) cells to induce their proliferation and activation into CC chemokine-activated killer (CHAK) cells 68. In addition, RANTES can act directly on CD8+ T cells to provoke biphasic mobilization of Ca2+, which is important for initiating chemotaxis and Ag-  12  Figure 1.4. Pre-existing RANTES mRNA in memory CD8+ T lymphocytes is translated during the recall response (adapted from Refs. 5, 6). (A) Memory CD8+ T cells contain high levels of spliced cytoplasmic RANTES mRNA that is translated only following the TCR signal. (B) Translation of the pre-existing RANTES mRNA is necessary for rapid secretion of high amount of RANTES protein by memory CD8+ T cells.  13  independent proliferation 69, as well as to increase IFN-γ production and to enhance cytolytic activity via upregulation of the Fas ligand 70. Notably, RANTES, together with the chemokines MIP-1α and MIP-1β, has been identified as a natural HIV-suppressive factor 71. Considering these important roles of RANTES in the immune system, understanding the mechanism of RANTES production could aid the development on novel medicines. 1.4.2. IFN-γ IFNs are potent antiviral and growth-inhibitory cytokines. The IFN family includes structurally related type I IFNs (IFN-α1, -α2, -α4, -α5, -α6, -α7, -α8, -α10, α13, -α14, -α16, -α17, -α21, IFN-β, IFN-δ, IFN-ε, IFN-κ, IFN-τ, and IFN-ω), a structurally distinct type II IFN (IFN-γ) (reviewed in Ref. 72), and novel class of type III IFNs (IFN-λ1, -λ2 and -λ3 that are also known as IL-29, IL-28A and IL-28B, respectively) 73, 74. IFN-γ is an important cytokine that has strong immunoregulatory properties and plays a key role in antiviral, antibacterial, and anti-tumor responses (reviewed in Refs. 75, 76, 77). IFN-γ is secreted by activated immune cells: mainly T cells and NK cells, but also (to a lesser extent) NKT cells and professional APCs (e.g. B cells) (reviewed in Ref. 78). This cytokine binds to the type II IFN receptor, which is composed of the IFNγR1 and IFNγR2 subunits (reviewed in Refs. 72, 78). Similarly to RANTES, the levels of IFN-γ mRNA are higher in memory CD8+ T lymphocytes than in naïve T cells 5, 7. Because resting memory CD8+ T lymphocytes do not synthesize IFN-γ protein (reviewed in Refs. 30, 33, 79, 80), the IFN-γ mRNA present in these cells may be translationally repressed. However, not all studies have detected stored  14  IFN-γ mRNA in memory T cells 6, 54, and the kinetics of RANTES and IFN-γ mRNA production are different following TCR stimulation 6. Remarkably, a recent study has identified large stores of translationally repressed IFN-γ mRNA in self-reactive CD8+ T lymphocytes that are unable to produce IFN-γ (anergic cells) 81, underlining the importance of IFN-γ translational control in CD8+ T cells. Because of the profound effects of IFN-γ on nearly every cell in the body, expression of this cytokine is tightly controlled (reviewed in Ref. 76). Even though the precise mechanism of IFN-γ post-transcriptional control is still under investigation, it has been reported that various receptor-induced activation signals prompt p38 mitogenactivated protein kinase (MAPK)- and/or signal transducer and activator of transcription (STAT) 4-dependent IFN-γ mRNA stabilization 82, 83, 84, 85. Moreover, IFN-γ mRNA stability is enhanced by IL-12 and IL-18 treatment, the process that is dependent on the 3′ untranslated region (3’UTR) of IFN-γ mRNA 86. IFN-γ mRNA stabilization in response to treatment with anti-lymphocyte function-associated antigen 1 (LFA-1) and anti-CD3 antibodies (Abs) depends on Hu protein R (HuR), a protein that binds 3’UTR of several cytokine mRNAs 87. Furthermore, IFN-γ signaling itself induces stabilization of numerous cytokine and chemokine mRNAs via direct association of IFNγR with the MyD88 adaptor protein 88. This mRNA stabilization requires signaling via p38 MAPK and mixed-lineage kinase (MLK) 3, and is dependent on AU-rich elements (AREs) in 3’UTR 88. Since IFN-γ affects CD8+ T lymphocytes directly 89, it is conceivable that IFNγ induces stabilization of its own mRNA via IFNγR-MyD88-p38 signaling axis. Furthermore, translational repression of the highly expressed IFN-γ mRNA in anergic CD8+ T lymphocytes is mediated by AREs in 3’UTR of IFN-γ mRNA 81. Based on the  15  above evidence, it is possible that IFN-γ mRNA synthesis in response to TCR-induced activation of naïve CD8+ T lymphocytes leads to the stabilization of IFN-γ mRNA, which is mediated by the interaction of HuR with AREs in 3’UTR of IFN-γ mRNA. Curiously, there is also evidence that IFN-γ mRNA is regulated by a pseudoknot formed by 14 nucleotides in its 5′UTR 90, 91 and by the microRNA (miRNA) miR-29 92. This tight control of IFN-γ production is necessary because of the strong effects of this cytokine on almost every cell type in the body (reviewed in Refs. 72, 75, 76, 78). Among its other effects, IFN-γ induces the development of TH1 cells and prevents the formation of TH2 cells (reviewed in Ref. 93), TH17 cells 94, and regulatory T (TReg) cells 95  . Notably, IFN-γ plays a significant role in the host immune response to cancer  (reviewed in Ref. 96). Furthermore, IFN-γ acts directly on CD4+ and CD8+ T lymphocytes to induce optimal effector cell expansion and survival 89, 97. In clinical settings, IFN-γ is used for treatment of chronic granulomatous disease 98, 99 and severe malignant osteopetrosis 99, 100. IFN-γ is also being evaluated in clinical trials for treatment of idiopathic pulmonary fibrosis, tuberculosis, AIDS, hepatitis C, and cancer (reviewed in Ref. 76). Considering the above benefits of IFN-γ, further understanding the mechanism of IFN-γ translational control and production by memory CD8+ T lymphocytes could assist the development on novel drugs and vaccines.  1.5. Kinetics of mRNA expression In a groundbreaking study from Dr. David Baltimore’s lab, the authors found that gene expression during an inflammatory response is regulated by a combination of mRNA stability and transcriptional control and can be grouped into 3 categories: (Group  16  I) genes whose mRNAs are rapidly expressed and mostly degraded, but low levels persist; (Group II) genes whose mRNAs are expressed almost as fast as Group I, but slowly decline with time; and (Group III) genes whose mRNAs are expressed at later timepoints and accumulate over time (Figure 1.5, A) 101. Note that IFN-γ mRNA belongs to Group I and RANTES mRNA belongs to Group III 6, 101. There are numerous genes that are highly expressed either only in effector and memory CD8+ T lymphocytes, or only in memory cells, but not in naïve cells (Figure 1.5, B) 7. It is probable that some of these highly expressed mRNAs are translationally repressed. Therefore, translational repression in memory CD8+ T lymphocytes could be a common phenomenon, underlining the importance of understanding it further.  1.6. MiRNAs and AREs can direct target mRNAs to P-bodies for storage and can facilitate subsequent reactivation of translation 1.6.1 P-bodies P-bodies are granular cytoplasmic aggregates that contain translationallyrepressed mRNAs 13, 102 in a complex with repressor 103 and/or mRNA turnoverpromoting proteins (reviewed in Refs. 104-106) (Figure 1.6). All eukaryotic cells examined to date in organisms ranging from yeast to plants to humans contain P-bodies (reviewed in Ref. 106). P-bodies are important for general mRNA decay, nonsense-mediated mRNA decay, ARE-mediated mRNA decay, and miRNA-induced mRNA silencing (reviewed in Refs. 104-106). However, in addition to mRNA degradation, P-bodies also support miRNAmediated translational repression (reviewed in Refs. 104-106) and mediate the storage of mRNAs in a translationally-repressed state that can re-enter the translational pool following various extracellular signals or metabolic triggers 107-109. P-bodies were 17  Figure 1.5. Kinetics of mRNA expression during an inflammatory response and during the development of memory CD8+ T lymphocytes (adapted from Refs. 7, 101). (A) A representative microarray analysis of gene expression following stimulation of NIH 3T3 fibroblasts with TNF-α for 0.5, 2 or 12 hours, and categorization of genes whose expression differs by a factor of ≥2.0 from time 0 into three kinetically distinct groups: Group I, Group II, and Group III. 101 (B) Expression of 431 genes in Ag-specific CD8+ T cells isolated 8, 15, 22, or 40 days post infection that differ by a factor of ≥1.7 from naïve cells in at least one of the time points. 7  18  Figure 1.6. MiRNAs are able to direct target mRNAs into P-bodies for storage and subsequent recruitment for translation (adapted from Ref. 110). Mature miRNAs repress translation of the target mRNA, and the miRNA-mRNA complex is transported into P-bodies for either storage or degradation. A specific signal can induce the stored mRNA to re-enter the translationally-active pool. GW182 and RCK/p54 are the P-body protein components necessary for miRNA-mediated translational repression, and DCP1a is important for mRNA decapping. The anti-GWB serum reacts with several P-body protein components, and can thus be used as a general P-body detection reagent.  19  originally identified as “mXRN1p granules” in 1997 111, but were later re-discovered as “GW-bodies” (GWBs) 112 and as “decapping-bodies” 113, 114 in 2002. Considering the role of P-bodies in reversible translational repression, I hypothesized that P-bodies in memory CD8+ T lymphocytes might be sites for the accumulation of translationally-repressed mRNAs that encode important effector molecules, such as RANTES and IFN-γ. Many different P-body protein components have been identified (reviewed in Ref. 106  ), but this study is focused on only three P-body markers that were chosen based on the  best available reagents for their detection and their distinct functions. GW182 is a major protein component of P-bodies 115 and is crucial for miRNA-induced silencing 116-118. RCK/p54 (also known as DDX6) is a general translation repressor protein that accumulates in P-bodies, is required for the function of some miRNAs, and is downregulated under certain cellular conditions to mediate recruitment of translationallyrepressed mRNAs back into the translationally-active pool 109, 118. DCP1a is a P-body component that is involved in mRNA decapping 119, 120. In addition to these markers of Pbodies, or potentially different subsets of P-bodies, the reference human anti-GWB serum 18033 that was used to (re)-discover P-bodies 112 reacts with several P-body components 121  and has been used as a general P-body reagent in this study. P-bodies, however, are not the only type of RNA granules. In fact, P-bodies share  some of their protein components with other cytoplasmic RNA granules, such as stress granules and dendritic P-bodies (reviewed in Refs. 105, 122, 123) (Figure 1.7). Understanding the specific roles of each type of RNA granule is an active area of research.  20  To my knowledge, P-bodies had not been characterized in lymphocytes at the time this study was initiated. However, during the course of this study, a report documenting RCK/p54 granules in activated CD4+ T cells was published 124. The authors found that Roquin, a novel ubiquitin ligase that is an essential negative regulator of follicular helper T (TFH) cells and autoantibody responses 125, co-localizes and directly interacts with RCK/p54 in cytoplasmic granules 124. Moreover, Roquin is necessary for the translational repression of mRNA encoding the inducible costimulator (ICOS) 124. This involves Roquin binding directly to the 3' UTR of ICOS mRNA in a miRNAindependent manner 124. As for RCK/p54, Roquin also localizes to stress granules 126. The fact that Roquin interacts with RCK/p54 124 and is necessary to prevent autoimmunity 125 by differentiating the specialized functions CD28 and ICOS co-stimulatory molecules 127, underlines the importance of characterizing the roles of RCK/p54 and other P-body components in lymphocytes. It is important to note that even though RCK/p54 has been detected in activated CD4+ T lymphocytes, it is currently unknown whether CD8+ T or B lymphocytes contain RCK/p54 granules. Also, it remains to be determined whether resting CD4+ T lymphocytes contain RCK/p54 granules, or whether any T or B lymphocytes contain P-body-specific markers. Because all eukaryotic cells examined to date contain P-bodies (reviewed in Ref. 106  ), the formation of P-bodies is a consequence of miRNA production 128, and certain  miRNAs are required for lymphocyte fate specification, development, and function (reviewed in Ref. 129), it is likely that lymphocytes contain P-bodies. However, there are no reports focused on characterization of P-bodies in lymphocytes, and this study provides the first thorough analysis of P-bodies in T and B lymphocytes.  21  Figure 1.7. P-bodies share some of their protein components with other RNA granules (adapted from Ref. 123). P-bodies and stress granules share some of the components and are both involved in translational repression, but stress granules only aggregate during stress. In addition, there are examples of RNA granules that share both P-body-specific and stress-granule-specific protein components, for example, dendritic P-bodies (reviewed in Refs. 105, 122, 123).  22  1.6.2 MiRNAs MiRNAs represent a class of small (21-23 nucleotides) non-coding RNAs that have recently emerged as master regulators of post-transcriptional gene expression that influence development and cell differentiation (reviewed in Refs. 110, 129-133). Upon binding to a specific site in the target mRNA, the miRNA represses translation, followed by either storage of the mRNA in P-bodies or degradation by the P-body mRNA decay machinery (reviewed in Ref. 110) (Figure 1.6). The expression of many cytokines is regulated by miRNAs 92, 134. However, even though the miRNA miR-146a controls RANTES production at the translational level, miR-146a does not target RANTES mRNA directly 135, and three different algorithms predicted that there are no miRNA binding sites in 3’UTR of RANTES mRNA 92. Still, as the prediction algorithms improve, it is possible that miRNAs targeting RANTES mRNA will be identified in the future. On the other hand, IFN-γ mRNA is predicted to be targeted by miR-29 in its 3’UTR 92. In addition, recent miRNA expression profiling studies revealed intrinsic differences between memory and naïve lymphocytes 136-139, and it is possible that some of these miRNAs are involved in translational control of cytokine production. It has been suggested that P-bodies act as storage sites for specific mRNAs that need to be quickly mobilized into polyribosomes in response to receptor signaling or changes in cellular metabolism 140. For example, the miRNA miR-122 represses translation of the cationic amino acid transporter 1 (CAT-1) mRNA in human hepatoma cells 140, but under certain stress conditions miR-122 facilitates the rapid release of CAT1 mRNA from P-bodies and subsequent recruitment into polyribosomes 140-142. Furthermore, depending on an external signal, miRNA activity can directly switch from  23  translational repression to activation 143. Another mechanism to recruit the translationally-repressed mRNA from P-bodies back into the translationally-active pool is to target P-body protein components. For example, during hypoxia, the miRNA miR-130 targets RCK/p54 mRNA, downregulating RCK/p54 and thereby facilitating translation of the previously repressed hypoxia inducible factor 1α (HIF-1α) mRNA and allowing the cell to overcome hypoxia 109. Therefore, it is possible that upon a TCR signal, memory CD8+ T lymphocyte-specific miRNAs trigger the release of stored effector mRNAs from P-bodies and facilitate translation, enabling the rapid recall response. 1.6.3 AREs AREs are AUUUA pentamers within U-rich regions in 3’UTR of mRNAs 144. Even though AREs are often referred to as decay elements 144, 145, AREs can also upregulate translation in response to extracellular stimuli 143, 146 (reviewed in Refs. 134, 147  ). In addition, not all AREs are the same, as the nonamer UUAUUUAUU is the ARE  motif minimally required for mRNA degradation. 148 ARE-binding proteins (ARE-BPs) mediate ARE functions and are classified into two groups: (1) decay promoting/destabilizing ARE-BPs, such as tristetraprolin (TTP) 149, butyrate response factor 1 (BRF1), AU-rich binding factor 1 (AUF1) 150, and KH-type splicing regulatory protein (KHSRP); and (2) stabilizing ARE-BPs, such as HuR 150-152 (reviewed in Refs. 134, 153, 154  ). Notably, AREs have been reported to direct mRNAs to P-bodies 155 (reviewed  in Ref. 106). Moreover, it was mentioned in other reports that 3’UTR of RANTES mRNA does not contain AREs 101, but 3’UTR of IFN-γ mRNA does 81, 101. Using ARESite (http://rna.tbi.univie.ac.at/cgi-bin/AREsite.cgi), the online resource for ARE detection and analysis, I have confirmed that 3’UTR of RANTES mRNA does not contain AREs  24  and found that 3’UTR of IFN-γ mRNA contains 6 AREs. Hence, it is conceivable that during the recall phase, TCR engagement in memory CD8+ T lymphocytes induces AREdependent recruitment of translationally-repressed effector mRNAs, such as IFN-γ mRNA, from P-bodies back into the translationally-active pool.  1.7. P-bodies move on microtubules and could be involved in localized translation Localized mRNA translation is an emerging field in biology, and it is now appreciated that RNA distribution at specific subcellular sites is a widespread property of polarized cells (reviewed in Refs. 156, 157). For example, localization of β-actin mRNA at the leading edge of migrating fibroblasts is important for persistent cell movement 158, 159. Similarly, apical localization of Crumbs mRNA is necessary establishing apicobasal polarity in Drosophila epithelial cells 160. Various mRNAs accumulate in protruding pseudopodia of migrating fibroblasts 161 and at spreading initiation centers that are formed by spreading fibroblasts 162. In addition, some mRNAs are actively transported on microtubules 160, 163 until being anchored at the correct destination 163. It has been proposed that P-bodies contribute to localized translation, since Pbody-like structures, called neuronal granules, deliver stored mRNAs to the correct site of translation in neurons, thus preventing premature protein synthesis and allowing dendrites and axons to extend in the right direction 164, 156, 165. Moreover, P-body movement on microtubules has been documented using live-cell imaging 11. The microtubule cytoskeleton polarizes towards the immune synapse that develops when a T lymphocyte, for example, binds an APC displaying peptide-MHC complexes that can engage the TCR and initiate TCR signaling 166-168. Polarization of the microtubule25  organizing center (MTOC) towards the immune synapse is necessary for directed secretion of effector cytokines (e.g. IL-4) 169. Recent work has shown that T cells secrete some effector proteins in a directional manner towards the immune synapse and the APC (e.g. IFN-γ), whereas other cytokines are secreted at random sites (e.g. TNF-α) 170. Given this information, I propose that mRNAs encoding effector molecules destined for targeted secretion, are stored in P-bodies of memory CD8+ T lymphocytes, and are transported towards the immune synapse, where these mRNAs can be mobilized into polyribosomes for localized translation and directed secretion towards the target cell, during the recall response.  1.8. NK cells have adaptive immune features due to an unknown mechanism The memory and the recall phases are normally ascribed only to cells of the adaptive immune system. However, these two phases have recently been documented in natural killer (NK) cells that are classified as members of the innate immune system 8-10. In contrast to their naïve counterparts, these memory-like NK cells have intracellular IFN-γ mRNA (based on analysis of Yeti mice), and secrete IFN-γ more rapidly than naïve NK cells upon reactivation 9, 10. Since no IFN-γ is synthesized by resting memory-like NK cells 9, 10, this IFN-γ mRNA could be stored in P-bodies and mobilized for translation during the recall response. Understanding the adaptive immune features of NK cells is an active area of research in immunology and could provide new insights into NK-cell function. My hypothesis is that storage of effector mRNAs in P-bodies is a mechanism shared by NK cells and T cells that facilitates rapid recall responses. However, the  26  models of immune memory are better defined for T cells, and therefore this study is focused on the role of P-bodies in memory CD8+ T lymphocytes.  1.9. Significance Addressing the questions proposed in this study will help improve current understanding of immunological memory. Vaccination is based on inducing a longlasting immune memory, and thus requires solid theoretical knowledge. Furthermore, a recent study demonstrated that a human miRNA directs HIV-1 RNA into P-bodies, and that this is crucial for suppressing HIV-1 replication 171. However, the experiments in that study involving P-bodies were done using HEK 293T cells, suggesting that there is a need to image P-bodies in T cells in order to understand defense mechanisms against viral infections. This project is the first to directly address the role of P-bodies in lymphocytes, and I hope it lays a foundation for diverse future studies.  1.10. Hypotheses and goals Overall hypothesis: RANTES and IFN-γ effector mRNAs are stored in P-bodies of memory CD8+ T lymphocytes and are directed for translation at the immune synapse during the recall response (Illustration 1.1). •  Hypothesis 1: T and B lymphocytes contain P-bodies. o Goal 1.1: Establish whether human and murine T and B-cell lines, as well as primary murine CD4+ T, CD8+ T, and B lymphocytes contain GWBs and express the P-body markers GW182, RCK/p54, and DCP1a that are localized to discrete cytoplasmic foci.  27  o Goal 1.2: Determine whether GWBs, GW182, RCK/p54, and DCP1a are localized to the same cytoplasmic granules. •  Hypothesis 2: Memory CD8+ T lymphocytes contain more P-bodies than naïve T cells, and P-bodies in memory cells store RANTES and IFN-γ mRNAs. o Goal 2.1: Compare total expression levels, cumulative area, and numbers of P-bodies in naïve, activated, effector, and memory CD8+ T lymphocytes. o Goal 2.2: Determine the localization of RANTES and IFN-γ mRNAs relative to P-bodies in naïve, activated, effector, and memory CD8+ T lymphocytes.  •  Hypothesis 3: Upon activation of a lymphocyte, P-bodies are recruited to the immune synapse, where effector mRNAs are released for translation. o Goal 3.1: Determine whether P-bodies are recruited to clustered IgM in primary murine B lymphocytes. o Goal 3.2: Test whether P-bodies are localized to the immune synapse in naïve, effector, and memory CD8+ T lymphocytes.  28  Illustration 1.1. Overall hypothesis. RANTES and IFN-γ effector mRNAs are stored in P-bodies of memory CD8+ T lymphocytes and are directed for translation at the immune synapse during the recall response.  29  CHAPTER 2: MATERIALS AND METHODS 2.1. Cell culture The HEK 293 human embryonic kidney cell line, T-cell lines (Jurkat, MOLT4, HBP-ALL, DO11.10, EL4, CTLL2), and B-cell lines (RAMOS, BJAB, A20, and WEHI231) were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). The K40B-1 pro-B cell line and the K40B-2 pre-B cell line were obtained from Dr. Anthony DeFranco (University of California, San Francisco, CA, USA), the OPM2 B-cell line was a gift from Dr. Alice Mui (Immunity and Infection Research Centre, Vancouver, BC), and the 5TGM1 B-cell line was a gift from Dr. Babatunde O. Oyajobi (University of Texas Health Science Center, San Antonio, TX, USA). HEK 293 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco, Carlsbad, CA, USA #12491) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco #12676), 2 mM L-glutamine, 1 mM sodium pyruvate, 50 µM ß-mercaptoethanol, 15 U/mL penicillin, and 50 µg/mL streptomycin (complete DMEM) in 5% CO2 at 37°C. All T-cell lines and B-cell lines were cultured in RPMI medium 1640 (Gibco #21870) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 µM ß-mercaptoethanol, 15 U/ml penicillin, and 50 µg/ml streptomycin (complete RPMI) in 5% CO2 at 37°C. CTLL2 is an IL-2-dependent 172 CD8+ T-cell line derived from a C57BL/6 mouse 173, 174, and therefore CTLL2 cells were cultured in complete RPMI with 5 ng/mL recombinant murine IL-2 (R&D Systems, Minneapolis, MN #402-ML). All cell lines were maintained in culture for ≤ 4 weeks.  30  2.2. Mice C57BL/6 (B6) and C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT-I; B6 background, transgenic for Vα2 Vβ5 TCR recognizing ovalbumin (OVA) peptide OVA257–264 SIINFEKL on H-2Kb) mice175 were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). All mice were used at 6–12 weeks of age. All animal experiments followed protocols approved by the Animal Care Committee of the University of British Columbia (Vancouver, BC).  2.3. Isolation of primary T and B lymphocytes Primary murine CD4+ and CD8+ T cells were purified from the spleens, as well as from axillary, superficial cervical, and inguinal lymph nodes, of B6 mice via negative selection using the automated system RoboSep (StemCell Technologies, Vancouver, Canada) and the mouse CD4+ and CD8+ T-cell enrichment kits (Stem Cell Technologies #19752R and #19753R, respectively) according to manufacturer’s instructions. Primary B cells were purified from spleens of B6 mice via negative selection using RoboSep and the mouse B-cell enrichment kit (Stem Cell Technologies #19714A). The purity of isolated T and B cells was analyzed each time by incubating the cells with 10 µg/mL of the Fc receptor blocking reagent α-mouse CD16/CD32 (clone 2.4G2, ATCC, prepared in house) mAb for 10 minutes on ice, and then staining with αB220-eFluor450 (clone RA36B2, eBioscience, San Diego, CA, USA #48-0452-82), αIgM-FITC (clone eB121-15F9, eBioscience #11-5890-85), αCD3e-PE (clone 145-2C11, BD Biosciences, Franklin Lakes, NJ, USA #553064), αCD4-PE-Cy7 (clone RM4-5, BD Biosciences #552775), αCD44-APC (clone IM7, BD Biosciences #559250), and αCD8a-APC-Cy7 (clone 5331  6.7, BD Biosciences #557654) mAbs for 30 minutes on ice while protected from light. Following staining, the cells were washed twice with FACS buffer [phosphate-buffered saline, pH 7.4 (PBS, Gibco #10010) plus 2% FBS]. The cells were then resuspended in 500 µL FACS buffer for analysis by flow cytometry. Data were acquired using a BD Biosciences LSR II flow cytometer and analyzed with FlowJo software (Tree Star, Ashland, OR, USA).  2.4. Immunoblot analysis of P-body protein expression Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (30 mM TrisHCl, pH 7.4, 150 mM NaCl, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EDTA, 1 mM PMSF, 10 µg/mL leupeptin, 1 µg/mL aprotinin, 1 mM Na3VO4, 25 mM βglycerophosphate, 1 µg/mL Pepstatin A). Bicinchoninic acid (BCA) assays were performed to measure protein concentration of each lysate. Cell extracts were subsequently loaded on 10% acrylamide gels at 40 µg/lane, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membranes. The membranes were incubated with 5% skim milk in Tris buffered saline, pH 7.4 (TBS) for 1 hour at room temperature (RT) and washed 3 times with TBS containing 0.1% Tween 20 (TBST) for 10 minutes. Immunoblotting was performed with human serum 18033 against GWBs (from Dr. Marvin Fritzler, University of Calgary, AB, 1:5000 dilution), with mouse mAb recognizing human and mouse GW182 (clone 4B6, Abcam, Cambridge, England #ab70522, 1:500 dilution), or with rabbit Abs against human and mouse RCK/p54 (Abcam #ab40684, 1:1000 dilution) or against DCP1a (Abcam #ab47811, 1:500 dilution). All Abs were diluted in TBS containing 5% bovine serum albumin, fraction V (BSA; Fisher Scientific #BP1600-100) and incubated 32  overnight at 4ºC. The membranes were then washed 3 times with TBST and probed with horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA #109-035-088, 1:5000 dilution), goat anti-mouse IgG (Bio-Rad Laboratories, Hercules, CA, USA #170-6516, 1:3000 dilution), or goat anti-rabbit IgG (Bio-Rad #170-6515, 1:5000 dilution), and immunoreactive bands were visualized using enhanced chemiluminescence (ECL). Equal loading of gel lanes was confirmed by re-probing the membranes with mouse mAb against human and mouse actin (clone C4, MP Biochemicals, Solon, OH, USA #691001, 1:500 dilution) and with a rabbit mAb against human and mouse α-tubulin (clone EP1332Y, Epitomics, Burlingame, CA, USA #1878-1, 1:5000 dilution).  2.5. Dual analysis of T and B lymphocytes by flow cytometry and confocal microscopy 2.5.1. Initial protocol development The protocol below briefly outlines the variations that have been tested in order to develop the final protocol that is described in detail in Chapter 3. CTLL2 cells were fixed with 4% paraformaldehyde (PFA, Electron Microscopy Sciences #15710) in PBS for 20 minutes at 37°C, followed by 1 minute on ice and a wash with FACS buffer. The cells were then permeabilized either with saponin wash buffer (SWB) containing 0.1-0.5% saponin (Sigma-Aldrich, St. Louis, MO, USA #S-2149), 0.1% NaN3 (Fisher Scientific International, Hampton, NH, USA #26628-22-8), and 0-1% normal goat serum (Jackson ImmunoResearch #005-000-121) in PBS, or with PBS containing 0.1-0.5% Triton X-100 (Sigma-Aldrich #X-100) for 10 minutes at RT. Next,  33  the cells were incubated with blocking buffer containing either 0.1-0.5% saponin or no saponin, 0.05-0.1% NaN3, 0-0.5% BSA, 0-2.5% goat serum, and 0-50% MAX block solution (Active Motif, Carlsbad, CA, USA #15252) in PBS for 3 hours at RT, and washed again with either SWB or PBS. Primary Ab staining was performed by incubating 106 cells with a mouse mAb against human and mouse α-tubulin (clone TU01, Abcam #ab7750, 1:250 dilution) and/or a rabbit Ab against human and mouse calnexin (Sigma-Aldrich #C4731, 1:200 dilution) in 100 µL Ab buffer (0.2% saponin or no saponin, 0.1% NaN3, 1% BSA, 0-5% goat serum in PBS) at 4ºC overnight. Matched concentrations of mouse IgG1 (eBioscience #14-4714-85) and rabbit IgG (Jackson Labs # 011-000-003) served as isotype controls for Abs against α-tubulin and calnexin, respectively. On the next day, the cells were washed 3 times with SWB or PBS and incubated with goat anti-mouse IgG conjugated to either Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA #A-11001 or #A-11029, 1:1000 dilution) or to Alexa Fluor 647 (Invitrogen #A-21235 or #A-21240, 1:1000 dilution), rhodamine-phalloidin (Invitrogen #R415, 1:100 dilution), and/or goat anti-rabbit IgG conjugated to either Alexa Fluor 488 (Invitrogen #A-11008, 1:1000 dilution) or to Alexa Fluor 647 (Invitrogen #A-21244, 1:1000 dilution) in 100 µL Ab buffer per 106 cells for 1 hour at RT while protected from light. The cells were subsequently washed twice with SWB or PBS, once with FACS buffer, and then resuspended in ~10-20 µL FACS buffer. For confocal microscopy, 2-10 µL of the cells was mixed with 2-10 µL of ProLong Gold antifade reagent with 4',6diamidino-2-phenylindole (DAPI) (Invitrogen # P36935) on a glass slide (Fisher Scientific #12-550-15), covered with an 18-mm square coverslip (coverslip; Fisher Scientific #12-542A), incubated at 4ºC overnight while protected from light, sealed with  34  a clear nail polish, and imaged on an Olympus IX81 confocal microscope (Shinjuku, Tokyo, Japan) using a 100x objective. Data were analyzed using FluoView 1000 software (Olympus). To analyze the same cells by flow cytometry, the remaining ~2-18 µL of cells was resuspended in 300-500 µL FACS buffer, analyzed using a BD Biosciences LSRII flow cytometer, and data were processed using FlowJo software. 2.5.2. Protocol adaptation for mRNA fluorescent in situ hybridization (FISH) FISH was initially performed exactly as described in Refs. 176, 177. The protocol was then adapted for staining in suspension and dual analysis by flow cytometry and confocal microscopy. CTLL2 cells were fixed with 4% PFA in PBS for 20 minutes at 37°C, followed by 1 minute on ice and a wash with FACS buffer or PBS. The cells were then permeabilized either with SWB or with PBS containing 0.1-0.5% Triton X-100 for 10 minutes at RT. Next, 106 cells were incubated with 100 µL blocking buffer containing either 0.1-0.5% saponin or no saponin, 0.05-0.1% NaN3, 0-0.5% BSA, 0-2.5% goat serum, and 0-50% MAX block solution in PBS for 3 hours at RT, and washed again with either SWB or PBS. Pre-hybridization was performed in either (1) 100 µL saponin prehybridization buffer containing 2x saline sodium citrate (SSC; 17.53 mg/mL NaCl, sodium citrate 8.82 mg/mL, pH 7.5), 20% formamide (Invitrogen #15515-026), 1 µg/µL yeast Saccharomyces cerevisiae tRNA (Invitrogen #15401-029) in Ab buffer, or (2) 100 µL of buffer containing 2x SSC, 20% formamide, 1 µg/µL yeast tRNA, and 0.2% BSA in PBS, for 15 minutes at 37°C. For mRNA FISH, the cells were incubated in 100 µL  35  hybridization buffer containing 5 pmol Cy3-oligo-d(T) probe (40 nt) specific for poly(A) mRNA in pre-hybridization buffer for 16 hours at 46°C while protected from light. On the next day, the cells were washed twice with 2x SSC and 20% formamide in either SWB or PBS for 5 minutes at 37°C, twice with 2x SSC in either SWB or PBS for 5 minutes at 37°C, once with 1x SSC in either SWB or PBS for 5 minutes at RT, and once with PBS for 5 minute at RT. For confocal microscopy, the cells were resuspended in ~10-20 µL FACS buffer, and 2-10 µL of cells was mixed with 2-10 µL of ProLong Gold antifade reagent with DAPI on a glass slide, covered with an 18-mm square coverslip, incubated at 4ºC overnight while protected from light, sealed with a clear nail polish, and imaged on an Olympus IX81 confocal microscope with FluoView 1000 software and a 100x objective. To analyze the same cells by flow cytometry, the remaining ~2-18 µL of cells was resuspended in 300-500 µL FACS buffer, examined on a BD Biosciences LSRII flow cytometer, and data were processed using FlowJo software. 2.5.3. Analysis on P-body markers in T and B lymphocytes Jurkat, HBP-ALL, DO11.10, CTLL2, RAMOS, and WEHI-231 cells, as well as primary CD4+ T, CD8+ T, and B cells from B6 mice were fixed with 4% PFA in PBS for 20 minutes at 37°C, followed by 1 minute on ice and a wash with FACS buffer. The cells were then permeabilized with SWB (0.1% saponin, 0.1% NaN3, and 1% normal goat serum in PBS) for 10 minutes at RT, incubated with blocking buffer (0.1% saponin, 0.05% NaN3, 0.5% BSA, 2.5% goat serum, and 50% MAX block solution in PBS) for 3 hours at RT, and washed again with SWB. For B-cell analysis, the cells were incubated with 10 µg/mL of the Fc receptor blocking reagent α-mouse CD16/CD32 (clone 2.4G2,  36  ATCC, prepared in house) mAb in Ab buffer (0.2% saponin, 0.1% NaN3, 1% BSA, 5% goat serum in PBS) for 20 minutes at RT, followed by a wash with SWB. Primary Ab staining was performed by incubating 106 cells with human serum against GWBs at a 1:500 dilution, with the mouse mAb against human and mouse GW182 at a 1:25 dilution, or with rabbit Abs against human and mouse RCK/p54 at a 1:250 dilution or DCP1a at a 1:100 dilution in 100 µL Ab buffer at 4ºC overnight. Matched concentrations of normal human serum (Jackson Labs, Los Gatos, CA, USA #009-000-001), mouse IgG1, and rabbit IgG served as isotype controls for GWBs, GW182, and RCK/p54 or DCP1a, respectively. On the next day, the cells were washed 3 times with SWB and incubated with secondary Ab master mixes containing goat anti-human IgG conjugated to Alexa Fluor 488 (Fcγ-specific, Invitrogen #H10120, 1:100 dilution), goat anti-mouse IgG conjugated to Alexa Fluor 488 (H+L-specific, highly cross-adsorbed, Invitrogen #A11029, 1:1000 dilution), or goat anti-rabbit IgG conjugated to Alexa Fluor 488 (H+Lspecific, Invitrogen #A-11008, 1:1000 dilution) plus rhodamine-phalloidin (1:100 dilution) in 100 µL Ab buffer per 106 cells for 1 hour at RT while protected from light. The cells were subsequently washed twice with SWB, once with FACS buffer, and then resuspended in ~12 µL FACS buffer. For confocal microscopy, 3 µL of cells was mixed with 4 µL of ProLong Gold antifade reagent with DAPI on a glass slide, covered with an 18-mm square coverslip, incubated at 4ºC overnight while protected from light, sealed with a clear nail polish, and imaged on an Olympus IX81 confocal microscope with FluoView 1000 software and a 100x objective. Quantification of cumulative area and number of P-bodies for each P-body marker was done using ImagePro Plus software (Media Cybernetics, Bethesda, MD, USA). To analyze the same cells by flow cytometry,  37  remaining ~9 µL of cells were resuspended in 350 µL FACS buffer, examined on a BD Biosciences LSRII flow cytometer, and data were processed using FlowJo software. For further details see Illustrations 4.1-4.3. 2.5.4. Co-localization analysis on P-body markers in T and B lymphocytes Staining of P-body markers in T and B cells was performed as described in section 2.5.3 with the following exceptions: (1) 1º Ab staining was done using every possible combination of two 1º Abs, (2) no rhodamine-phalloidin was added to the 2º Ab master mixes, and (3) in addition to an Alexa Fluor 488-conjugated 2º Ab, another 2º Ab conjugated to Alexa Fluor 647 and specific for either rabbit IgG (Invitrogen #A-21244, 1:1000 dilution) or mouse IgG1 (Invitrogen #A-21240, 1:1000 dilution) was added to the 2º master mixes. Quantification of Pearson’s correlation coefficient, as well as M1 and M2 overlap coefficients, for each P-body marker was done using ImagePro Plus software.  2.6. Analysis of P-bodies in primary B lymphocytes following activation 2.6.1. Activation of primary B lymphocytes with αIgM and/or αCD40 and IL-4 B cells were isolated from spleens of B6 mice as described in section 2.3 and were resuspended to 107 cells/mL in FACS buffer. To initiate the timecourse, 1 mL complete RPMI containing (1) 10 µg/mL F(ab’)2 donkey α-mouse IgM (µ chain-specific, Jackson ImmunoResearch #715-006-020), (2) 5 µg/mL rat α-mouse CD40 mAb (clone 1C10, prepared in-house), 5 ng/mL recombinant mouse IL-4 (R&D Systems #404-ML010), and αIgM as above, or (3) αCD40 and IL-4 as above, was added to 106 cells in 5  38  mL round-bottom tissue culture (TC) tubes with caps. The cells were then gently mixed and incubated for 20 minutes, 1 hour, 6 hours, or 24 hours at 37ºC in 5% CO2. At the end of the incubation period, the cells were gently resuspended in 1 mL PBS, fixed by addition of 1 mL 8% PFA in PBS, incubated for 20 minutes at 37°C, followed by 1 minute on ice, washed with FACS buffer, and stored in Flow buffer (PBS, 2% FBS, 0.1% NaN3) until staining. For additional details see Illustration 4.4. 2.6.2. Analysis of activation markers on B lymphocytes treated with αIgM and/or αCD40 and IL-4 Activation markers were analyzed simultaneously using 5 x 105 fixed B cells that were (1) not activated, (2) activated with αIgM, (3) activated with αIgM, αCD40, and IL-4, or (4) activated with αCD40 and IL-4 for 20 minutes, 1 hour, 6 hours, or 24 hours using the same master mixes for all relevant samples. The cells were incubated for 10 minutes on ice with 10 µg/mL of the Fc receptor blocking reagent α-mouse CD16/CD32 (clone 2.4G2, ATCC, prepared in house) mAb, and then stained with αB220-eFluor450 (clone RA3-6B2, eBioscience #48-0452-82), αIgM-FITC (clone eB121-15F9, eBioscience #11-5890-85), αCD80-PE (clone 16-10A1, eBioscience #12-0801-82), αCD69-PE-Cy7 (clone H1.2F3, eBioscience #25-0691-82), αCD86-APC (clone GL1, eBioscience #17-0862-82) mAbs for 30 minutes on ice while protected from light. Following staining, the cells were washed twice with FACS buffer and then resuspended in 500 µL FACS buffer for analysis by flow cytometry. Data were acquired using a BD Biosciences LSR II flow cytometer and analyzed with FlowJo software.  39  2.6.3. Analysis of P-body markers in activated B lymphocytes P-body markers were analyzed simultaneously as described in section 2.5.3 using fixed B cells that were (1) not activated, (2) activated with αIgM, (3) activated with αIgM, αCD40, and IL-4, or (4) activated with αCD40 and IL-4 for 20 minutes, 1 hour, 6 hours, or 24 hours using master mixes of either 1º Abs or 2º Abs for all samples. In addition, the cells were also stained with 2 µg/mL goat anti-mouse IgM conjugated to Alexa Fluor 647 (µ chain-specific, Invitrogen #A21238, 1:1000 dilution) in order to visualize clustered IgM.  2.7. Differentiation of naïve OT-I CD8+ T lymphocytes into effector and memory CD8+ T lymphocytes Effector and memory OT-I CD8+ T cells were generated in vitro according to G.M. Griffith’s lab 178 and T.H. Watts’ lab 52 protocols, respectively (with modifications suggested by Amy Saunders and Nina Maeshima from P. Johnson lab at UBC), as summarized in Illustration 5.1. These procedures are described in detail below. 2.7.1 Differentiating bone marrow-derived dendritic cells On day 1, bone marrow (BM) cells were isolated from the femurs of B6 mice, counted, and 2 x 106 BM cells were plated into 10 mL DC medium [complete RPMI medium supplemented with 20 ng/mL recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF; PeproTech, Rocky Hill, NJ, USA #315-03)] in 10cm Optilux plates (BD Falcon #351005) to keep the DCs in suspension. On day 4, another 10 mL of DC medium was added to each plate. On day 7, 10 mL of medium was gently removed from each plate and replaced with 10 mL of fresh DC medium. On day 8, 40  >70% of the non-adherent and loosely adherent cells were CD11c+ immature bone marrow-derived dendritic cells (BMDCs). Note that is important to use pyrogen-free sterile filter tips for handling DCs and all reagents in order to avoid contamination with lipopolysaccharide (LPS), which would cause premature DC activation. This protocol was received from Dr. Ken Harder’s lab and taught to me by Munreet Chehal (University of British Columbia). 2.7.2. Maturing BMDCs and loading with the OVA257-264 peptide SIINFEKL Immature BMDCs were harvested, counted, and 2 x 106 BMDCs were plated into 10 mL DC medium with 100 ng/mL LPS from Escherichia coli O111:B4 (Sigma-Aldrich #L2630) in 10-cm TC-treated plates (BD Falcon # 353003) overnight. On the next day, the medium was removed and adherent cells (now mature BMDCs) were washed with PBS, covered with 10 mL DC medium containing 1 µM OVA257-264 peptide SIINFEKL (Cedarlane Laboratories, Burlington, ON #RP10611-1MG), and incubated at 37ºC in 5% CO2 for 3 hours. 2.7.3. Isolating naïve CD8+ T lymphocytes from OT-I mice Naïve (CD44Low) CD8+ T cells were purified from the spleens, as well as from axillary, superficial cervical, and inguinal lymph nodes, of OT-I mice via negative selection using the automated RoboSep system and the CD8+ T-cell enrichment kit according to manufacturer’s instructions. In order to remove naturally occurring memoryphenotype (CD44High) CD8+ T cells via the RoboSep αbiotin mAb-coated magnetic beads, an αCD44-biotin mAb (clone IM7, eBioscience #13-­‐‑0441-85) was added at 1 µg/mL immediately before starting the RoboSep cycle. The cells were then counted and 41  resuspended to 107 cells/mL in FACS buffer following isolation. Naïve cells required for intracellular staining were gently resuspended in 1 mL PBS, fixed by addition of 1 mL 8% PFA in PBS and incubation for 20 minutes at 37°C, followed by 1 minute on ice, washed with FACS buffer, and stored in Flow buffer until staining. 2.7.4. Activating naïve OT-I CD8+ T lymphocytes with SIINFEKL-loaded mature BMDCs On day 0 of activation (d0), DC medium containing SIINFEKL was gently removed from the mature BMDCs, and the cells were washed with PBS and covered with 10 mL complete RPMI. Next, 2 x 106 naïve OT-I CD8+ T cells were added to SIINFEKL-loaded mature BMDCs (1:1 T cells:BMDC ratio) and cultured in presence of 5 ng/mL recombinant murine IL-2 (R&D Systems #402-ML) at 37ºC in 5% CO2 for 48 hours. On day 2 of activation (d2), non-adherent cells, which included activated CD8+ T cells, were harvested, counted, and resuspended to 107 cells/mL in FACS buffer. Activated cells required for intracellular staining were gently resuspended in 1 mL PBS, fixed by addition of 1 mL 8% PFA in PBS and incubation for 20 minutes at 37°C, followed by 1 minute on ice, washed with FACS buffer, and stored in Flow buffer until staining. 2.7.5. Differentiating activated OT-I CD8+ T lymphocytes into effector CD8+ T lymphocytes On day 2 of activation (d2), 106 activated CD8+ T cells were plated into a T175 flask with 80 mL complete RPMI medium containing 5 ng/mL IL-2 and cultured for an additional 48 hours. On day 4 of activation (d4), live lymphocytes were isolated from dead cells and debris using Lympholyte-M (Cedarlane #CL5031) according to the  42  manufacturer’s instructions, plated into a new T175 flask with 80 mL complete RPMI medium containing 5 ng/mL IL-2, and cultured for another 24 hours. On day 5 of activation (d5), non-adherent cells were harvested as effector CD8+ T cells, counted, and resuspended to 107 cells/mL in FACS buffer. Effector cells required for intracellular staining were gently resuspended in 1 mL PBS, fixed by addition of 1 mL 8% PFA in PBS and incubation for 20 minutes at 37°C, followed by 1 minute on ice, washed with FACS buffer, and stored in Flow buffer until staining. 2.7.6. Differentiating activated OT-I CD8+ T lymphocytes into memory CD8+ T lymphocytes On day 2 of activation (d2), 106 activated CD8+ T cells were plated into a T175 flask with 80 mL complete RPMI medium containing 20 ng/mL recombinant murine IL15 (R&D Systems #447-ML) and cultured for an additional 48 hours. On day 4 of activation (d4), live lymphocytes were isolated from dead cells and debris using Lympholyte-M according to the manufacturer’s instructions, plated into a new T175 flask with 80 mL complete RPMI medium containing 20 ng/mL IL-15, and cultured for another 7 days, with the medium being changed every 2-3 days to accommodate cell growth. On day 11 of activation (d11), live lymphocytes were again separated from dead cells and debris using Lympholyte-M, plated into a new T175 flask with 80 mL complete RPMI medium containing 20 ng/mL IL-15, and cultured for another 24 hours. On day 12 of activation (d12), non-adherent cells were harvested as memory CD8+ T cells, counted, and resuspended to 107 cells/mL in FACS buffer. Memory cells required for intracellular staining were gently resuspended in 1 mL PBS, fixed by addition of 1 mL 8% PFA in PBS and incubation for 20 minutes at 37°C, followed by 1 minute on ice, washed with  43  FACS buffer, and stored in Flow buffer until staining.  2.8. Phenotypic and functional analysis of naïve, activated, effector, and memory OT-I CD8+ T lymphocytes 2.8.1. Analysis of surface markers on naïve, activated, effector, and memory OT-I CD8+ T lymphocytes by flow cytometry Surface marker analysis was performed by incubating 5 x 105 naïve (before and after negative selection), activated, effector, or memory CD8+ T cells with 10 µg/mL of the Fc receptor blocking reagent α-mouse CD16/CD32 (clone 2.4G2, ATCC, prepared in house) mAb for 10 minutes on ice, and then staining with (1) αCD3e-PB (clone 500A2, BD Biosciences #558214), αCD25-FITC (clone 7D4, BD Biosciences #553071), αCD122-PE (clone 5H4, eBioscience 12-1221-81), αCD62L-PE-Cy7 (clone MEL-14, BD Biosciences #560516), αCD44-APC (clone IM7, BD Biosciences #559250), and αCD8a-APC-Cy7 (clone 53-6.7, BD Biosciences #557654) mAbs, (2) αCD3e-PB, αCD43-FITC (clone eBioR2/60, eBioscience #11-0431-81), αCD27-PE (clone LG.7F9, eBioscience #12-0271-81), αCD69-PE-Cy7 (clone H1.2F3, eBioscience #25-0691-82), αCD43 (activated isoform)-AlexaFluor649 (clone 1B11, Ab Lab, University of British Columbia, Vancouver, BC #AB10FY01MW174), and αCD8a-APC-Cy7 mAbs, or (3) αCD3e-PB, αCD127-PE-Cy5 (clone A7R34, eBioscience #15-1271-81), αKLRG1-APC (clone 2F1, eBioscience #17-5893-81), and αCD8a-APC-Cy7 mAbs for 30 minutes on ice while protected from light. Following staining, the cells were washed twice with FACS buffer and resuspended in 500 µL FACS buffer for analysis on an LSR II flow  44  cytometer using identical settings for all cell types analyzed over multiple days within each experiment. Data were analyzed with FlowJo software. 2.8.2. Analysis of proliferation potential of naïve OT-I CD8+ T lymphocytes activated with mature SIINFEKL-loaded BMDCs by CFSE dilution assay Immature BMDCs were prepared as described in sections 2.7.1, and 4 x 105 cells were seeded into each well of a 6-well TC plate containing 2 mL DC medium. These cells were then activated with LPS and loaded with the OVA257-264 peptide SIINFEKL as described in section 2.7.2. Naïve CD8+ T cells were isolated from OT-I mice as described in section 2.7.3 and resuspend to 107 cells/mL in FACS buffer. To label these cells with carboxyfluorescein succinimidyl ester (CFSE), the cells were washed with 10 mL PBS, resuspended to 107 cells/mL in 2 µM carboxyfluorescein diacetate succinimidyl ester (CFDA-SE; Invitrogen #C1157), and incubated for 8 minutes at 37ºC while protected from light. This incubation allows a non-fluorescent and the highly cell permeable molecule CFDA-SE to enter the cell cytoplasm, where intracellular esterases remove acetate groups from CFDA-SE converting the molecule into fluorescent CFSE, which is retained within cells because it is covalently coupled to intracellular molecules and is weakly cell permeable 179. The CFSE labeling reaction was stopped by adding an equal volume of FBS. The cells were then washed with 10 mL PBS, re-counted, and resuspended to 107 cells/mL in FACS buffer. To start the CFSE dilution assay, 4 x 105 CFSE-labeled naïve OT-I CD8+ T cells were added to 4 x 105 SIINFEKL-loaded mature BMDCs (1:1 T cell:BMDC ratio) in 2  45  mL complete RPMI medium and cultured in the presence of 5 ng/mL IL-2 at 37ºC in 5% CO2 for 22, 28, 34, or 48 hours while protected from light. At the end of the incubation period, the cells were resuspended in FACS buffer containing 1 µg/mL 7-AAD (Calbiochem #129935), incubated for 10 minutes on ice while protected from light, and analyzed on an LSR II flow cytometer. Data were processed using FlowJo software Proliferation Platform to analyze live OT-I CD8+ T cells (CFSE+ 7-AAD-). 2.8.3. Analysis of cytotoxic potential in effector and memory OT-I CD8+ T lymphocytes activated with SIINFEKL-loaded EL4 target cells by cytotoxicity assay To be used as target cells, EL4 cells (murine H-2b-expressing T cells from C57BL/6 lymphoma) were harvested, washed with serum-free RPMI medium, counted, resuspended to 106 cells/mL in serum-free RPMI medium containing 1 µM CellTracker Orange (Invitrogen #C2927), and incubated at 37ºC in 5% CO2 for 30 minutes while protected from light. To stop the labeling reaction, FBS was added to 10% of the final volume, and the cells were washed with complete RPMI medium. To load the CellTracker Orange-labeled EL4 cells with the OVA257-264 peptide SIINFEKL, the cells were then resuspended in complete RPMI medium containing 1 µM SIINFEKL and incubated at 37ºC in 5% CO2 for 3 hours while protected from light. During this incubation period, naïve CD8+ T cells were isolated from OT-I mice as described in section 2.7.3 to serve as a control for cytotoxicity assay. In addition, effector or memory OT-I CD8+ T cells were harvested from in vitro culture flasks and resuspended to 107 cells/mL in FACS buffer.  46  To start the cytotoxicity assay, CellTracker Orange-labeled SIINFEKL-loaded EL4 cells were resuspend to 4 x 106 cells/mL in fresh complete RPMI medium, and 105 cells were aliquoted into wells of a 96-well round-bottom TC plate (BD Falcon #353917). Next, naïve and either effector or memory OT-I CD8+ T cells were added to the EL4 cells at effector to target ratios (E : T) of 10, 3, 1, 0.3, 0.1 in triplicate. The final volume was brought up to 275 µL by adding complete RPMI medium, and the co-cultures were incubated at 37ºC in 5% CO2 for 4 hours while protected from light. To analyze longterm cytotoxic potential, 3 x 104 OT-I CD8+ T cells were added to 105 EL4 cells (E : T = 0.3) in wells of a 24-well TC plate (BD Falcon #353935), the final volume was brought up to 2 mL by adding complete RPMI medium with or without 5 ng/mL IL-2, and the cocultures were incubated at 37ºC in 5% CO2 for 24 or 48 hours while protected from light. At the end of the incubation period, the cells were resuspended in FACS buffer containing 1 µg/mL 7-AAD, incubated for 10 minutes on ice while protected from light, and analyzed on an LSR II flow cytometer. Data were processed using FlowJo software. Specific cytotoxicity was calculated by subtracting the percent of dead EL4 cells (CellTracker Orange+ 7-AAD+) incubated without OT-I CD8+ T cells from the percent of dead EL4 cells that had been incubated with OT-I CD8+ T cells for each condition. For further details see Illustrations 5.2 and 5.3. 2.8.4. Analysis of proliferation potential in effector and memory OT-I CD8+ T lymphocytes activated with SIINFEKL-loaded EL4 target cells by CFSE dilution assay EL4 cells were labeled with CellTracker Orange and loaded with the OVA257-264 peptide SIINFEKL as described in section 2.8.3. Naïve CD8+ T cells were isolated from  47  OT-I mice as described in section 2.7.3 to serve as a control for the CFSE dilution assay. Effector or memory OT-I CD8+ T cells were harvested from in vitro culture flasks and resuspend cells to 107 cells/mL in FACS buffer. Next, naïve and either effector or memory OT-I CD8+ T cells were labeled with CFSE as described in section 2.8.2. To start the CFSE dilution assay, 3 x 104 CFSE-labeled naïve and either effector or memory OT-I CD8+ T cells were added to 105 CellTracker Orange-labeled SIINFEKL-loaded EL4 cells (E : T = 0.3) in wells of a 24-well TC plate, the final volume was brought up to 2 mL by adding complete RPMI medium with or without 5 ng/mL IL2, and the co-cultures were incubated at 37ºC in 5% CO2 for 24 or 48 hours while protected from light. At the end of the incubation period, the cells were resuspended in FACS buffer containing 1 µg/mL 7-AAD, incubated for 10 minutes on ice while protected from light, and analyzed on an LSR II flow cytometer. Data were processed using FlowJo software Proliferation Platform to analyze live OT-I CD8+ T cells (CFSE+ CellTracker Orange- 7AAD-). For further details see Illustration 5.3. 2.8.5. Analysis of survival potential in effector and memory OT-I CD8+ T lymphocytes activated with SIINFEKL-loaded EL4 target cells As described in section 2.8.4, 3 x 104 CFSE-labeled naïve and either effector or memory OT-I CD8+ T cells were co-cultured with 105 CellTracker Orange-labeled SIINFEKL-loaded EL4 cells (E : T = 0.3) in wells of a 24-well TC plate containing 2 mL complete RPMI medium with or without 5 ng/mL IL-2 at 37ºC in 5% CO2 for 24 or 48 hours while protected from light.  48  At the end of the incubation period, the cells were resuspended in FACS buffer containing 1 µg/mL 7-AAD, incubated for 10 minutes on ice while protected from light, and analyzed on an LSR II flow cytometer. Data were processed using FlowJo software. The percent of surviving OT-I CD8+ T cells was calculated by subtracting the percent of dead OT-I CD8+ T cells (CFSE+ CellTracker Orange- 7-AAD+) from 100%. For additional details see Illustration 5.3.  2.9. Analysis of P-body markers and mRNA localization to P-bodies in naïve, activated, effector, and memory OT-I CD8+ T lymphocytes 2.9.1. Dual analysis of P-body markers in naïve, activated, effector, and memory OT-I CD8+ T lymphocytes by flow cytometry and confocal microscopy P-body markers were analyzed simultaneously for fixed naïve, activated, effector, and memory OT-I CD8+ T cells as described in section 2.5.3 with the following exception: instead of rhodamine-phalloidin, the cells were stained with either (1) a mouse mAb against human and mouse α-tubulin (clone TU-01, Abcam #ab7750, 1:250 dilution) and goat secondary Ab against mouse IgG1 conjugated to Alexa Fluor 647 (Invitrogen #A-21240, 1:1000 dilution), or (2) a rabbit mAb against human and mouse α-tubulin (clone EP1332Y, Epitomics, Burlingame, CA, USA #1878-1, 1:500 dilution) and goat secondary Ab against rabbit IgG conjugated to Alexa Fluor 647 (Invitrogen #A-21244, 1:1000 dilution). 2.9.2. Poly(A) mRNA FISH and dual analysis of naïve, activated, effector, and memory OT-I CD8+ T lymphocytes by flow cytometry and confocal microscopy Poly(A) mRNA FISH was performed simultaneously for fixed naïve, activated, effector, and memory OT-I CD8+ T cells according to the final protocol derived from  49  section 2.5.2. The cells were fixed with 4% PFA in PBS for 20 minutes at 37°C, followed by 1 minute on ice and a wash with FACS buffer. The cells were then permeabilized with SWB for 10 minutes at RT. Next, 106 cells were incubated with 100 µL blocking buffer containing 50% MAX block solution in Ab buffer for 3 hours at RT, and washed again with SWB. Pre-hybridization was performed in 100 µL pre-hybridization buffer containing 2x SSC, 20% formamide, and 1 µg/µL yeast tRNA in Ab buffer for 15 minutes at 37°C. For poly(A) mRNA FISH, the cells were incubated in 100 µL hybridization buffer containing 5 pmol Cy3-oligo-d(T) probe (40 nt) specific for poly(A) mRNA in pre-hybridization buffer for 16 hours at 46°C while protected from light. On the next day, the cells were washed twice with SWB containing 2x SSC and 20% formamide for 5 minutes at 37°C, twice with SWB containing 2x SSC for 5 minutes at 37°C, once with SWB containing 1x SSC for 5 minutes at RT, and once with PBS for 5 minute at RT. For confocal microscopy, the cells were resuspended in ~12 µL Flow buffer, and 3 µL of cells was mixed with 4 µL of ProLong Gold antifade reagent with DAPI on a glass slide, covered with an 18-mm square coverslip, incubated at 4ºC overnight while protected from light, sealed with a clear nail polish, and imaged on an Olympus IX81 confocal microscope with FluoView 1000 software and a 100x objective. To analyze the same cells by flow cytometry, the remaining ~9 µL of cells were resuspended in 350 µL FACS buffer, examined on a BD Biosciences LSRII flow cytometer, and data were processed using FlowJo software.  50  2.9.3. RANTES mRNA FISH and dual analysis of naïve, activated, effector, and memory OT-I CD8+ T lymphocytes by flow cytometry and confocal microscopy RANTES mRNA FISH was performed simultaneously for fixed naïve, activated, effector, and memory OT-I CD8+ T cells according to the protocol derived from section 2.9.2 and Ref. 180. Fixed cells were dehydrated with 70% ethanol for 5 minutes at RT, and then with 100% ethanol at 4ºC overnight. On the next day, the cells were rehydrated with PBS and permeabilized with saponin FISH buffer (SFB; 0.2% saponin, and 0.1% NaN3 in PBS) for 10 minutes at RT. Pre-hybridization was performed in 100 µL pre-hybridization buffer containing 2x SSC and 40% formamide in SFB for 15 minutes at 37°C. At this time, 1 pmol of locked nucleic acid (LNA) probes, which are more stable than RNA probes, labeled with digoxigenin (DIG) on both ends and specific for either murine RANTES mRNA (5’-DIG-CCCTCTATCCTAGCTCATCTCC-DIG-3’, Exiqon, Vedbaek, Denmark #300500-15) or a scramble control (5’-DIGGTGTAACACGTCTATACGCCCA-DIG-3’, Exiqon #300514-15) were diluted in 40 µL probe buffer containing 80% formamide, 125 µg/mL yeast tRNA, and 125 µg/mL salmon sperm DNA (Sigma-Aldrich #D9156) in PBS, heated to 95ºC for 5 minutes, and transferred to 37ºC water bath for 5 minutes. Next, the cells were resuspended in 40 µL hybridization buffer containing 20% dextran sulfate (dextran sulfate sodium salt from Leuconostoc spp., Mw > 500,000, Sigma #D8906-5G), 0.2% BSA, and 4x SSC in SFB, warmed to 37ºC, and mixed with the 40 µL probe prepared earlier. Hybridization was performed for 3 hours at 37°C. Following hybridization, the cells were washed once with post-hybridization buffer 1 (2x SSC and 40% formamide in SFB) for 5 minutes at 37°C, twice with post-hybridization buffer 2 (1x SSC and 40% formamide in SFB) for 5  51  minutes at 37°C, once with post-hybridization buffer 3 (1x SSC in SWB) for 10 minutes at RT, and once with SWB. In order to detect DIG, the cells were incubated in 100 µL Ab buffer containing 2 µg/mL α-DIG-rhodamine sheep IgG Fab fragments (Hoffmann-La Roche, Basel, Switzerland #11297750910) for 1 hour at RT, washed twice with SWB, and three times with FACS buffer. For confocal microscopy, the cells were resuspended in ~12 µL Flow buffer, and 3 µL of cells was mixed with 4 µL of ProLong Gold antifade reagent with DAPI on a glass slide, covered with an 18-mm square coverslip, incubated at 4ºC overnight while protected from light, sealed with a clear nail polish, and imaged on an Olympus IX81 confocal microscope with FluoView 1000 software and a 100x objective. To analyze the same cells by flow cytometry, the remaining ~9 µL of cells were resuspended in 350 µL FACS buffer, examined on a BD Biosciences LSRII flow cytometer, and data were processed using FlowJo software. 2.9.4. FISH for dual analysis of IFN-γ mRNA in naïve, activated, effector, and memory OT-I CD8+ T lymphocytes by flow cytometry and confocal microscopy IFN-γ mRNA FISH was performed simultaneously for fixed naïve, activated, effector, and memory OT-I CD8+ T cells as described in section 2.9.3 with the following exception: instead of RANTES mRNA probe, a custom designed IFN-γ mRNA probe was used (5’-DIG-TCTGAGGTAGAAAGAGATAAT-DIG-3’, Exiqon #300500). 2.9.5 Analysis of RANTES and IFN-γ mRNA localization to P-bodies in naïve, activated, effector, and memory OT-I CD8+ T lymphocytes Fixed naïve, activated, effector, and memory OT-I CD8+ T cells were first immunostained with 1º Abs against P-body markers as described in section 2.5.3. 52  Immunostained cells were then fixed with 2% PFA in PBS for 10 min at RT, as suggested in Ref. 180. Next, RANTES or IFN-γ mRNA FISH was performed as described in sections 2.9.3 and 2.9.4 with the following modification: in addition to α-DIGrhodamine, the 2º Ab master mixes for staining cells also contained the 2º Abs that recognize the P-body marker 1º Abs as described in section 2.5.3.  2.10. Analysis of P-body localization in naïve OT-I CD8+ T lymphocytes during immune synapse formation on mature SIINFEKL-loaded BMDCs For immune synapse formation, 104 immature BMDCs were prepared as described in section 2.7.1 and plated into each well of µ-Slide 8-well ibiTreat microscopy chamber (Ibidi, München, Germany #80826) in 200 µL DC medium with 100 ng/mL LPS overnight. On the next day, the medium was removed and adherent cells (mature BMDCs) were washed with PBS, covered with 200 µL DC medium containing 1 µg/mL SIINFEKL, incubated at 37ºC in 5% CO2 for 3 hours, and washed again with PBS. Next, 2 x 104 naïve OT-I CD8+ T cells (isolated as described in section 2.7.3) in 10 µL FACS buffer were immediately added to with SIINFEKL-loaded mature BMDCs (2:1 T cell:BMDC ratio), incubated for 5 minutes at 37ºC in 5% CO2, covered with 200 µL complete RPMI medium containing 5 ng/mL IL-2, and cultured at 37ºC in 5% CO2 until 20 minutes, 1 hour, or 4 hours elapsed from co-culture initiation. At the end of the incubation period, the medium was gently removed from all wells in the chamber, and the cells were fixed by gently adding 200 µL 4% PFA in PBS, followed by an incubation for 20 minutes at 37ºC and for 5 minutes at 4ºC. The cells were then washed with Flow buffer and stored in 200 µL Flow buffer at 4ºC until permeabilization and staining.  53  To visualize immune synapses by confocal microscopy, a staining protocol was adapted from Ref. 178. Briefly, cell conjugates were permeabilized with 250 µL ice-cold methanol for 5 minutes at RT, washed 6 times with 250 µL PBS, incubated with 100 µL MAXblock reagent for 3 hours at RT, washed with 250 µL Wash buffer (0.1% NaN3 and 0.2% BSA in PBS), incubated with 1º Abs in 100 µL Wash buffer overnight at 4ºC as described in Table 2.1, washed 4 times with 250 µL Wash buffer for 5 minutes at RT, incubated with 2º Abs in 100 µL Wash buffer for 40 minutes at RT while protected from light as described in Table 2.1, washed again 4 times with 250 µL Wash buffer for 5 minutes at RT, and prepared for imaging by adding 100 µL mounting medium with DAPI and incubating for 10 minutes at 4°C. The chambers were stored up to 48 hours at 4°C while protected from light until being imaged on an Olympus IX81 confocal microscope with FluoView 1000 software and a 100x objective.  2.11. Analysis of P-body localization in naïve OT-I CD8+ T lymphocytes during the first asymmetric division after contacting mature SIINFEKL-loaded BMDCs To analyze the first asymmetric division, 2 x 104 naïve OT-I CD8+ T cells were incubated with 104 SIINFEKL-loaded mature BMDCs (2:1 T cell:BMDC ratio) as described in section 2.10. After 28 hours of co-culture, the medium was gently removed from all wells in the µ-Slide 8-well ibiTreat microscopy chamber, and the cells were fixed by gently adding 200 µL 4% PFA in PBS, followed by an incubation for 20 minutes at 37ºC and for 5 minutes at 4ºC. The cells were then washed with Flow buffer and stored in 200 µL Flow buffer at 4ºC until permeabilization and staining.  54  Table 2.1. Staining of immune synapses formed by naïve OT-I CD8+ T lymphocytes with SIINFEKL-loaded mature BMDCs for 20 minutes, 1 hour, and 4 hours. Well # 1  2  3  4  1°Ab specificity GWBs Lck αTub CD3e αTub GW182 RCK/p54 Lck DCP1a Lck -  1°Ab isotype Human IgG Mouse IgG2b Rabbit IgG Hamster IgG1 Rabbit IgG Mouse IgG1 Rabbit IgG Mouse IgG2b Rabbit IgG Mouse IgG2b -  1°Ab DF 500 100 250 50 250 25 250 100 100 100 -  2°Ab GtαHuIgG-Alexa488 GtαMsIgG-Alexa568 GtαRbIgG-Alexa647 FITC-conjugated 1º Ab GtαRbIgG-Alexa568 GtαMsIgG1-Alexa647 GtαRbIgG-Alexa488 GtαMsIgG-Alexa568 GtαRbIgG-Alexa488 GtαMsIgG-Alexa568 -  2°Ab DF 100 500 500 500 250 500 500 500 500 -  1º and 2º Abs specific for P-body markers are described in sections 2.5.3-2.5.4. Other Abs include: mouse anti-Lck (clone 3A5, Millipore, Billerica, MA, USA #05-435), hamster anti-CD3e-FITC (clone 145-2C11, BD Pharmingen #553061), rabbit anti-α-tubulin (Epitomics #1878-1), goat anti-mouse IgG-Alexa Fluor 568 (Invitrogen #A-11004), and goat anti-rabbit IgG-Alexa Fluor 568 (Invitrogen #A-11036). The final volume in each well was 100 µL. Note, αTub = α-tubulin, Ms = mouse, Rb = rabbit, Gt = goat, DF = dilution factor, V = volume, Alexa = Alexa Fluor.  55  To visualize the first asymmetric division by confocal microscopy, cell conjugates were permeabilized with 250 µL SWB for 5 minutes at RT, incubated with 100 µL MAXblock reagent in 100 µL Ab buffer for 3 hours at RT, washed with 250 µL SWB, incubated with 1º Abs in 100 µL Ab buffer overnight at 4ºC as described in Table 2.2, washed 4 times with 250 µL SWB for 5 minutes at RT, incubated with 2º Abs in 100 µL Ab buffer for 1 hour at RT while protected from light as described in Table 2.2, washed 2 times with 250 µL SWB for 5 minutes at RT, washed 2 time with 250 µL Flow buffer for 5 minutes at RT, and prepared for imaging by adding 100 µL mounting medium with DAPI and incubating for 10 minutes at 4°C. The chambers were stored for up to 48 hours at 4°C while protected from light until being imaged on an Olympus IX81 confocal microscope with FluoView 1000 software and a 100x objective.  2.12. Analysis of P-body localization in effector and memory OT-I CD8+ T lymphocytes during immune synapse formation on mature SIINFEKL-loaded EL4 target cells Effector and memory OT-I CD8+ T lymphocytes were prepared as described in sections 2.7.5 and 2.7.6, respectively. EL4 target cells were loaded with the OVA257-264 peptide SIINFEKL as described in section 2.8.2. For analysis of immune synapse formation, 2 x 104 SIINFEKL-loaded EL4 target cells were plated into each well of µSlide 8-well ibiTreat microscopy chamber in 200 µL complete RPMI medium and incubated for 30 minutes at 37ºC in 5% CO2, inducing EL4 cells to firmly adhere to the well bottom. Next, the medium was removed from the EL4 cells, and 4 x 104 effector or memory OT-I CD8+ T cells in 20 µL FACS buffer were added to SIINFEKL-loaded EL4 cells (2:1 T cell:EL4 cell ratio). The cells were then incubated for 5 minutes at 37ºC  56  Table 2.2. Staining the first asymmetric division of naïve OT-I CD8+ T lymphocytes on SIINFEKL-loaded mature BMDCs following 28 hours of co-culture. Well # 1  2  3  4  5  6  Stained for GWBs Par6 αTub GWBs Scribble αTub GW182 Par6 Scribble αTub GW182 Scribble RCK/p54 αTub Scribble DCP1a αTub  1°Ab isotype Human IgG Rabbit IgG Mouse IgG1 Human IgG Rabbit IgG Mouse IgG1 Mouse IgG1 Rabbit IgG Mouse IgM Rabbit IgG Mouse IgG1 Mouse IgM Rabbit IgG Mouse IgG1 Mouse IgM Rabbit IgG Mouse IgG1  1°Ab DF 500 250 100 500 50 100 25 250 25 250 25 25 250 100 25 100 100  2°Ab GtαHuIgG-Alexa488 GtαRbIgG-Alexa568 GtαMsIgG1-Alexa647 GtαHuIgG-Alexa488 GtαRbIgG-Alexa568 GtαMsIgG1-Alexa647 GtαMsIgG-Alexa488 GtαRbIgG-Alexa568 GtαMsIgM-Alexa488 GtαRbIgG-Alexa568 GtαMsIgG1-Alexa647 GtαMsIgM-Alexa488 GtαRbIgG-Alexa568 GtαMsIgG1-Alexa647 GtαMsIgM-Alexa488 GtαRbIgG-Alexa568 GtαMsIgG1-Alexa647  2°Ab DF 100 500 500 100 500 500 500 500 500 500 250 500 500 500 500 500 500  1º and 2º Abs specific for P-body markers are described in sections 2.5.3-2.5.4. Other Abs include: rabbit anti-Par6 (Abcam # ab45394), mouse anti-Scribble (clone D-8, Santa Cruz Biotechnology, Santa Cruz, CA #sc-55532), rabbit anti-Scribble (clone H-300, Santa Cruz Biotechnology # sc-28737), mouse anti-αtubulin (clone TU-01, Abcam #ab7750), rabbit anti-α-tubulin (Epitomics #1878-1), goat α-mouse IgMAlexa Fluor 488 (µ chain-specific, Invitrogen # A-21042), and goat anti-rabbit IgG-Alexa Fluor 568 (Invitrogen #A-11036). The final volume in each well was 100 µL. Note, αTub = α-tubulin, Ms = mouse, Rb = rabbit, Gt = goat, DF = dilution factor, V = volume, Alexa = Alexa Fluor.  57  in 5% CO2, before adding 200 µL complete RPMI medium containing 5 ng/mL IL-2, and culturing the cells at 37ºC in 5% CO2 until 20 minutes, 1 hour, or 4 hours elapsed from co-culture initiation. At the end of the incubation period, the medium was gently removed from all wells in the chamber, and the cells were fixed by gently adding 200 µL 4% PFA in PBS, followed by an incubation for 20 minutes at 37ºC and for 5 minutes at 4ºC. The cells were then washed with Flow buffer and stored in 200 µL Flow buffer at 4ºC until permeabilization and staining. Conjugates were stained and analyzed as described in section 2.10.  2.13. Statistics Student’s unpaired two-tailed t test was used to compare sets of unmatched samples. Student’s paired two-tailed t test was used to compare sets of matched samples.  58  CHAPTER 3: IMPROVED METHODOLOGY FOR STAINING LYMPHOCYTES IN SUSPENSION  3.1. Introduction In order to analyze the level and intracellular localization of a protein of interest using a small number of lymphocytes, I developed a staining protocol that allows dual analysis by flow cytometry and confocal microscopy to be performed by staining one sample of cells. Furthermore, I have adapted a fluorescent in situ hybridization (FISH) protocol 171 for this dual analysis. This protocol is especially useful for analysis of P-body components and mRNAs in primary T and B lymphocytes. Lymphocytes are immune cells that have an important role in protecting the body against pathogens. Resting T and B lymphocytes are non-adherent spherical cells that patrol the body in search for their cognate antigen and grow in suspension in culture. Lymphocytes are often plated on coverslips coated with poly-L-lysine, extracellular matrix, or antibodies for analysis by confocal microscopy 181, 182. However, the contact with these materials induces lymphocytes to change their shape (e.g. spreading) and to initiate various signaling pathways (e.g. integrin activation). These changes have the potential to affect the results of the study. Confocal microscopy is designed for analysis of relatively small cell numbers, and it is not always clear how representative is the sample analyzed. In contrast, flow cytometry can provide information about thousands of cells in a matter of seconds, and since all events are reported, one can make a conclusion if the population is homogeneous, or if sub-populations exist.  59  Here, I describe a method how to prepare lymphocytes for dual analysis by confocal microscopy and flow cytometry by fixing and staining them in suspension. In the example provided on applying this protocol, I describe how to stain murine CD8+ Tcell line CTLL2 in a 96-well plate format with Abs to the endoplasmic reticulum (ER) marker calnexin and to the microtubule (MT) component α-tubulin, as well as with phalloidin to mark filamentous actin (F-actin). I separate out ~25% cells for staining with DAPI and imaging by confocal microscopy, and then analyze the remaining ~75% cells by flow cytometry to determine staining consistency and expression of proteins of interest. This technique proved to be a reliable method to analyze fine intracellular features, as well as overall protein expression levels and staining consistency of lymphocytes in their native shape. The protocol could be useful to immunologists interested in various aspects of lymphocyte biology (e.g. cytoplasmic vs. nuclear location of NF-kB, nuclear subcompartmentalization, or localization of specific cytoplasmic proteins relative to Ag receptor). The method described here is applicable for other T and B-cell lines, and well as primary T and B lymphocytes. Finally, dual analysis is optional, and this protocol can be used for analysis by either flow cytometry or confocal microscopy.  60  3.2. Reagents, solutions, and equipment 3.2.1. Specific reagents and equipment Table 3.1. List of specific reagents and equipment. Antibody α-hu/ms calnexin α-hu/ms α-tubulin Mouse IgG1 isotype Rabbit IgG isotype α-rabbit IgG (H+L)AlexaFluor488 α-mouse IgG1AlexaFluor647  Company Sigma Abcam eBioscience Jackson Labs Invitrogen Invitrogen  Name of reagent RPMI medium 1640 Fetal bovine serum (FBS) Recombinant murine interleukin2 (IL-2) Phosphate-buffered saline pH 7.4 (PBS) Saponin Sodium azide (NaN3) Normal goat serum Bovine serum albumin, fraction V (BSA) 5 mL tubes with caps 16% paraformaldehyde (PFA) MAXblock blocking solution 96-well round-bottom TC plate Phalloidin-AlexaFluor488 Rhodamine-phalloidin Phalloidin-AlexaFluor647 18-mm square coverslips (coverslip) ProLong Gold antifade reagent with DAPI Microscope glass slides Fisherbrand Superfrost/Plus Flow cytometer Confocal microscope  Antibodies Catalogue # C4731 ab7750 14-4714-85 011-000-003 A11008 A21240  Clone Polyclonal TU-01 Polyclonal Rabbit IgG Polyclonal  Isotype Rabbit IgG Mouse IgG1 Mouse IgG1, κ Polyclonal Goat IgG  [Stock] 1.0 mg/mL 1.0 mg/mL 0.5 mg/mL 11.1 mg/mL 2.0 mg/mL  Polyclonal  Goat IgG  2.0 mg/mL  Other reagents Company Catalogue # Gibco 21870 Gibco 12676 R&D Systems 402-ML Gibco  10010  Sigma Fisher Scientific  S-2149 26628-22-8  Jackson ImmunoResearch Fisher Scientific  005-000-121  BD Falcon Electron Microscopy Sciences Active Motif BD Falcon Invitrogen Invitrogen Invitrogen Fisher Scientific  352054 15710 15252 353917 A12379 R415 A22287 12-542A  Invitrogen  P36935  Fisher Scientific  12-550-15  Equipment BD Bioscience Olympus  Comments Heat-inactivated at 56*C? Reconstituted to 10 µg/mL in PBS + 0.1% BSA  Make 10% NaN3 solution 1.0 g NaN3 in 10 mL ddH2O  9048-46-8  LSRII IX81  Sterile Electron microscopy grade  Mounting medium  FlowJo software (Tree Star) for data analysis FluoView 1000 software, 100x objective  61  3.2.2. Solution recipes Table 3.2. Recipe for FACS buffer. FACS Buffer (PBS, 2% FBS): 250 mL FBS PBS Filter-sterilize & store at 4°C  5 mL 245 mL  Table 3.3. Recipe for Flow buffer. Flow Buffer (PBS, 2% FBS, 0.1% NaN3): 250 mL FBS 10% NaN3 PBS Filter-sterilize & store at 4°C  5 mL 2.5 mL 242.5 mL  Table 3.4. Recipe for saponin wash buffer (SWB). Saponin Wash Buffer (SWB; PBS, 0.1% saponin, 0.1% NaN3, 1% goat serum): 250 mL Saponin (0.1% = 1 mg/mL) 250 mg 10% NaN3 2.5 mL Normal goat serum 2.5 mL PBS 245 mL Filter-sterilize & store at 4°C  Table 3.5. Recipe for antibody buffer (Ab buffer). Antibody Buffer (Ab buffer; PBS, 0.2% saponin, 0.1% NaN3, 1% BSA, 5% goat serum): 50 mL Saponin (0.2% = 2 mg/mL) 100 mg 10% NaN3 0.5 mL BSA 500 mg Normal goat serum 2.5 mL PBS 47 mL Filter-sterilize & store at 4°C  Table 3.6. Recipe for complete RPMI medium. Complete RPMI medium (RPMI medium 1640, 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 15 U/mL penicillin, 50 µg/mL streptomycin, and 50 µM ß-mercaptoethanol): 500 mL FBS 50 mL L-glutamine (200 mM) 5 mL Sodium pyruvate (100 mM) 5 mL Penicillin + streptomycin 2.5 mL (3 kU/mL penicillin & 10 mg/mL streptomycin) ß-mercaptoethanol (50 mM) 0.5 mL RPMI medium 1640 500 mL Store at 4°C  62  3.3. Protocol The experimental design for the analysis of calnexin and α-tubulin in CTLL2 cells is presented in Illustration 3.1 as an example of the protocol for dual analysis of lymphocytes by flow cytometry and confocal microscopy.  Illustration 3.1. Method outline for dual analysis of calnexin and α-tubulin in murine CD8+ T-cell line CTLL2 by flow cytometry and confocal microscopy. CTLL2 cells were fixed in 4% PFA, washed with FACS buffer, permeabilized with SWB, incubated with blocking buffer, stained in suspension with 1º Abs specific for calnexin and α-tubulin, and then with 2º Abs conjugated to Alexa Fluor 488 (calnexin, green) and to Alexa Fluor 647 (α-tubulin, violet) in combination with rhodamine-phalloidin (red) to mark F-actin. ~25% of the cells were injected into mounting medium with DAPI (blue) to visualize intracellular protein localization by confocal microscopy, and the remaining ~75% of the cells were analyzed by flow cytometry to determine staining consistency and expression of both proteins.  63  3.3.1. Day 1: Passage cells and make solutions CTLL2 is an IL-2-dependent 172 CD8+ T-cell line derived from a C57BL/6 mouse 173, 174  . CTLL2 cells can be cultured in RPMI medium 1640 supplemented with 10% heat-  inactivated FBS, 5 ng/mL IL-2, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 µM βmercaptoethanol, 15 U/ml penicillin, and 50 µg/ml streptomycin (complete RPMI medium, Table 3.6) in 5% CO2 at 37°C. CTLL2 cells grow in suspension, and should be split 2-3 times per week. Culturing the cells longer than 4 weeks is not recommended. 1) Passage CTLL2 cells. Make sure to have enough cells for the experiment. *Samples: calnexin+α-tubulin, isotype (2); flow cytometry controls: phalloidinAlexa488, rhodamine-phalloidin, phalloidin-647, Unstained (4); 1 extra. Therefore, need 106 cells/sample x 7 samples = 7 x 106 cells. 2) Make FACS buffer (Table 3.2), Flow buffer (Table 3.3), saponin wash buffer (SWB; Table 3.4), and antibody buffer (Ab buffer; Table 3.5). These buffers can be stored at 4°C and used for multiple experiments. 3.3.2. Day 2: Fix cells and stain with 1º Abs 1) Resuspend cells to 2 x 107 cells/mL in cold PBS 1.1) Spin cells down 6 minutes at 250 g, 4°C, decant supernatant. 1.2) Resuspend cells in 10 mL cold PBS, take out 100 µL cells to count, spin remaining cells down 6 minutes at 250 g, 4°C.  64  1.3) Count live cells using hemocytometer: resuspend 100 µL cells in 400 µL PBS [dilution factor (DF) = 5], take out 10 µL cells and resuspend in 10 µL trypan blue (DF = 2), then inject 10 µL into hemocytometer and count live cells in 4 squares. 1.4) Resuspend cells to 2 x 107 in cold PBS. Table 3.7. Sample calculation for counting cells before fixation. Sample CTLL2  VCells (mL) 10  DF 5 x 2 = 10  Cells in 4 Fields 80  :4 x DF x 104 (cells/mL) 2.0 x 106  Total cells 2.0 x 107  VPBS for 2 x 107 cells/mL 1.0 mL  1.5) Transfer the required number of cells (700 µL = 7 x 106 cells) into a 5 mL tube on ice. 2) Fix cells with 4% PFA 2.1) Make equal volume of 8% PFA in PBS (need 700 µL + 100 µL extra: dilute 400 µL 16% PFA in 400 µL PBS). 2.2) Fix cells by slowly adding 8% PFA (700 µL) and gently resuspend. 2.3) Incubate cells 20 minutes at 37°C, and then 1 minute on ice. 2.4) Spin cells down 5 minutes at 300 g, 4°C, decant supernatant. 2.5) Resuspend cells to 107 cells/mL in cold Flow buffer (700 µL = 7 x 106 cells). Note, these cells can be stored overnight at 4°C to continue on the next day. 3) Permeabilize and block cells 3.1) Spin cells down 5 minutes at 300 g, 4°C, decant supernatant.  65  3.2) Wash cells with SWB to permeabilize cells: resuspend cells in 700 µL of SWB, spin cells down 5 minutes at 300 g, 4°C, and decant supernatant. Note, it is recommended to use a minimum of 100 µL/106 cells of buffers for washing and incubation steps. 3.3) Make blocking buffer (50% Ab buffer, 50% MAXblock solution): need 100 µL/106 cells x 7 x 106 cells + 100 µL extra = 800 µL. Therefore, make 800 µL blocking buffer by mixing 400 µL Ab buffer and 400 µL MAXblock solution. 3.4) Resuspend cells in 700 µL of blocking buffer to prevent non-specific binding of Abs. 3.5) Incubate 3 hours at RT. 3.6) Spin cells down 5 minutes at 300 g, 4°C, decant supernatant. 3.7) Wash cells with SWB. 4) Stain cells with 1°Abs 4.1) Resuspend cells in 700 µL Ab buffer and aliquot 100 µL (106 cells)/well into appropriate wells in a 96-well round-bottom plate. Note, it is recommended to use a 96-well plate for staining more then 10 samples, whereas fewer samples can be stained in separate 5 mL tubes with caps. Here, I use 96-well plate for the purpose of demonstration, because most experiments have more than 20 samples.  66  Table 3.8. Sample plate outline for staining cells in suspension. Well # ->  1 Unstained  2 PhalloidinAlexa488  3 Rhodaminephalloidin  4 PhalloidinAlexa647  5 Calnexin + α-Tubulin  6 Calnexin + α-Tubulin Isotype  CTLL2 Flow cytometry controls  Samples  4.2) Add 1°Ab to each sample and resuspend. Table 3.9. Sample calculation for staining cells in suspension with primary antibodies. 1°Ab Calnexin α-tubulin Rabbit IgG 1/10 (calnexin isotype) Mouse IgG1 (α-tubulin isotype) Flow cytometry controls  [Stock] 1.0 mg/mL 1.0 mg/mL 1.11 mg/mL  DF 200 250 222  [Final] 5 µg/mL 4 µg/mL 5 µg/mL  Sample # 1 1 1  V Ab/sample 0.5 µL 0.4 µL 0.45 µL  V Ab Total 0.5 µL 0.4 µL 0.45 µL  0.5 mg/mL  125  4 µg/mL  1  0.8 µL  0.8 µL  -  -  -  4  -  -  4.3) Incubate plate at 4°C overnight. Note, avoid agitating the plate to prevent mixing between different wells. 3.3.3. Day 3: Stain cells with 2° Abs, mount onto slides, and analyze by flow cytometry 1) Stain cells with 2°Abs 1.1) Add 100 µL/well SWB and resuspend, spin plate down 5 minutes at 300 g, 4°C, and shake off supernatant. 1.2) Wash wells 3x with SWB: resuspend cells in 150 µL/well SWB, spin plate down 5 minutes at 300 g, 4°C, and shake off supernatant.  67  1.3) Make 2° Combo Mix: need 100 µL/well x 2 wells + 50 µL extra = 250 µL. Table 3.10. Sample calculation for staining cells in suspension with secondary antibodies and rhodamine-phalloidin. Reagent Goat α-rabbit IgG-Alexa488 Goat α-mouse IgG1-Alexa647 Rhodamine-phalloidin Ab buffer  [Stock] 2.0 mg/mL 2.0 mg/mL -  DF 1000 1000 100 -  [Final] 2.0 µg/mL 2.0 µg/mL -  V per 250 µL 0.25 µL 0.25 µL 2.5 µL 397 µL  1.4) Resuspend samples in 100 µL/well 2° Combo Mix. For flow cytometry controls, resuspend in 100 µL/well Ab buffer, and then add 1 µL phalloidin-Alexa Fluor 488, rhodamine-phalloidin, or phalloidin-Alexa Fluor 647 to appropriate wells and resuspend again. 1.5) Cover the plate with aluminum foil to protect from light, and incubate 1 hour at RT. Avoid agitating the plate. 1.6) Add 100 µL/well SWB and resuspend, spin plate down 5 minutes at 300 g, 4°C, and shake off supernatant. 1.7) Wash wells 2x with SWB, and then wash 1x with FACS buffer. 2) Mount cells for confocal microscopy 2.1) Resuspend cells in 150 µL/well Flow buffer and transfer into a 5 mL tube, wash well with additional 150 µL/well Flow buffer and add into the tube, spin cells down 5 minutes at 300 g, 4°C. 2.2) Carefully remove supernatant with a P200 pipettor. Remaining cells occupy ~10-15 µL.  68  2.3) Mount coverslip: resuspend cells using P20 pipettor set at 10 µL, place 4 µL of mounting medium with DAPI onto a glass slide, inject 3 µL cells into the mounting medium and resuspend, cover with a coverslip. 2.4) Let coverslip cure 24 hours at 4°C in the dark. 3) Analyze cells by flow cytometry 3.1) Resuspend remaining cells in 350 µL Flow buffer, and analyze on LSRII flow cytometer. Use single-stained flow cytometry controls to set up compensation gates. 3.2) Analyze data using FlowJo software. 3.3.4. Day 4: Seal coverslips and image by confocal microscopy 1) Seal coverslip with clear nail polish and leave to dry for 10 min at RT in the dark. Store coverslip at 4°C until imaging by confocal microscopy if less then 3 days, or store at -20°C long-term. The fluorophores are stable for at least 6 months. 2) Image coverslip with an Olympus IX81/FluoView FV1000 confocal microscope. We recommend a 100x oil objective for imaging intracellular features of lymphocytes.  3.4. Representative Results Flow cytometry results (Figure 3.1A) demonstrate that staining of the ER marker calnexin, the microtubule component α-tubulin, and F-actin in murine CD8+ T-cell line CTTL2 has some variation, but is mostly uniform. Confocal microscopy analysis suggests that the morphology of the marked structures is correct, confirming that the Abs  69  Figure 3.1. Dual analysis of lymphocytes by flow cytometry and confocal microscopy allows to visualize fine intracellular details, document consistency of staining, and determine protein expression levels. CTLL2 cells were fixed in 4% paraformaldehyde and stained in suspension with anti-calnexin + anti-rabbit IgG-Alexa Fluor 488, rhodamine-phalloidin (to mark F-actin), and anti-α-tubulin + anti-mouse IgG1-Alexa Fluor 647. Rabbit IgG and mouse IgG1 were used for isotype control staining of calnexin and α-tubulin, respectively. (A) ~25% of the cells were injected into mounting medium with DAPI and visualized by confocal microscopy using a 100x objective. (B) The remaining ~75% of the cells were analyzed by flow cytometry to assess staining on a population level. Note that F-actin staining demonstrates that the samples received the same amount of the secondary antibody master mix. Staining of calnexin or α-tubulin alone produced similar results. Bar: 5 µm.  70  identified calnexin and α-tubulin appropriately (Figure 3.1B). Comparison to isotype staining for both flow cytometry and confocal microscopy indicates that CTLL2 express calnexin and α-tubulin. Finally, F-actin staining in Figure 3.1A confirms that both “stained” and “isotype” samples received the same master mix.  3.5. Discussion 3.5.1. Critical steps and possible modifications •  Staining cells in single tubes vs. a 96-well plate format work equally well.  •  Storing cells in Flow buffer (FACS buffer containing 0.1% NaN3) protects samples from microbial growth, allowing one to permeabilize and stain the cells days or weeks later. Stained samples must be analyzed immediately or several hours later, because fluorescence intensity declines over time.  •  Fixation should be done with 4% PFA, because other methods (e.g. methanol) do not work well for this protocol.  •  Triton X-100 detergent should not be used for permeabilizing cells in suspension, because at low concentrations (e.g. 0.1%) Triton X-100 does not permeabilize cells, and at high concentrations (e.g. 0.5%) Triton X-100 ruptures the cells. Saponin works well for permeabilizing cells in suspension, but should only be used at concentrations 0.1-0.2%, because at higher concentrations (e.g. 0.5%) saponin makes large holes in cells. Note that saponin should be present in all buffers throughout the staining procedure.  •  Blocking with goat serum, BSA, and MAXblock reduces background and provides excellent signal-to-noise ratio improvement for both flow cytometry and  71  confocal microscopy analyses. Note that goat serum must be present in all buffers following blocking until the mounting step. •  It is recommended to use serum of the 2°Ab species for blocking. Hence, since goat 2°Abs were used here, I included goat serum into SWB and Ab buffer. However, depending on the 2°Ab used, the recipe of the buffers should be adjusted. Using the serum of the 1°Ab species will result in high background.  •  If possible, use cross-adsorbed or isotype specific Abs. For example, instead of a regular α-mouse IgG (H+L) Ab conjugated to Alexa Fluor 488 (Invitrogen #A11001), using a highly cross-adsorbed Ab (Invitrogen #A-11029) might reduce background. Similarly, instead of a α-mouse IgG (H+L)-Alexa Fluor 647 Ab (Invitrogen #A-21235), α-mouse IgG1 Ab (A-21240) could give better results.  •  Cell nuclei can be stained with DAPI or Hoechst before separating samples for analysis by flow cytometry and confocal microscopy. Alternatively, nuclear staining can be omitted completely, enabling the use of 405 nm laser for other markers.  •  I have successfully used this protocol for staining various T-cell and B-cell lines, as well as primary T- and B-lymphocytes. However, if working with B cells, it is critical to block their Fc receptors (FcRs) to avoid non-specific binding of Abs. This can be done by incubating the B cells with the αFcR blocking mAb 2.4G2 used at 25 µg/mL for 20 min at RT before adding 1°Abs. In addition, care should be taken to select Abs that would not bind to the B-cell antigen receptors.  72  •  Primary cells are small, and I recommend using at least 2 x 106 cells/sample if possible for your study. However, you should be able to obtain acceptable results even with ~1-2 x 105 cells/sample.  •  Imaging cells by confocal microscopy allows taking 3D stacks using optical slicing, and subsequent 3D-reconstruction. Optical slicing with 0.5 µm increments enables capturing fine detains of cell structure.  •  Since dual analysis is optional, this protocol can be used for analysis of lymphocytes by either flow cytometry or confocal microscopy.  •  I have successfully adapted this protocol for fluorescent in situ hybridization (FISH) (Figure 3.2, see section 2.5.2 for detains).  3.5.2. Conclusions •  Using this procedure, lymphocytes are fixed in suspension and retain their native morphology. In addition, signaling initiated by contacting a coated coverslip does not occur.  •  A single experiment with 2 detection methods reduces variability, saves time and reagents.  •  Information on staining consistency and protein expression levels is rapidly acquired using flow cytometry analysis, which provides more quantitative data than confocal microscopy.  •  Optical slicing allows much better resolution of intracellular features than image cytometry (e.g. ImageStreamX), which is aimed at providing cell images during flow cytometry.  73  Figure 3.2. Dual analysis of lymphocytes by flow cytometry and confocal microscopy can be adapted for FISH. CTLL2 cells were fixed in 4% paraformaldehyde and stained in suspension with Cy3-oligo-dT to label poly(A) mRNA. (A) ~25% of the cells were injected into mounting medium with DAPI and visualized by confocal microscopy using a 100x objective. (B) The remaining ~75% of the cells were analyzed by flow cytometry to demonstrate staining on a population level. Bar: 5 µm.  74  CHAPTER 4: ANALYSIS OF P-BODIES IN T AND B LYMPHOCYTES 4.1. Introduction Overall hypothesis: RANTES and IFN-γ effector mRNAs are stored in P-bodies of memory CD8+ T lymphocytes and are directed for translation at the immune synapse during the recall response (Illustration 1.1). •  Hypothesis 1: T and B lymphocytes contain P-bodies. o Goal 1.1: Establish whether human and murine T and B-cell lines, as well as primary murine CD4+ T, CD8+ T, and B lymphocytes contain GWBs and express the P-body markers GW182, RCK/p54, and DCP1a that are localized to discrete cytoplasmic foci. o Goal 1.2: Determine whether GWBs, GW182, RCK/p54, and DCP1a are localized to the same cytoplasmic granules.  •  Hypothesis 3: Upon activation of a lymphocyte, P-bodies are recruited to the immune synapse, where effector mRNAs are released for translation. o Goal 3.1: Determine whether P-bodies are recruited to clustered IgM in primary murine B lymphocytes. P-bodies have been detected in all eukaryotic cells tested to date (reviewed in Ref.  106  ), and thus it is likely that lymphocytes contain P-bodies. However, to my knowledge  there were no reports focused on characterizing P-bodies in lymphocytes. Therefore, in order to assess the overall hypothesis, my first task was to address hypothesis 1 and determine whether T and B lymphocytes contain P-bodies. In this chapter, I demonstrate  75  that T and B lymphocytes contain P-bodies and report localization, number, and colocalization of GWBs and the P-body markers GW182, RCK/p54, and DCP1a. Furthermore, I report that P-body markers are downregulated immediately following activation of primary murine B lymphocytes, and that the remaining P-body markers are localized in the proximity of clustered BCRs. Finally, I show that all P-body markers are upregulate in primary murine B lymphocytes 24 hours following BCR engagement. In order to address hypothesis 1, I have validated the Abs against the P-body protein components GW182, RCK/p54, and DCP1a, as well as the staining procedures, using HEK 293 cells that were previously shown to contain P-bodies 171, 183, 184. I have successfully detected GW182, RCK/p54, and DCP1a in HEK 293 lysates by western immunoblotting and observed GW182, RCK/p54, and DCP1a as cytoplasmic granules in HEK293 cells by confocal microscopy (data not shown). Furthermore, the numbers of Pbodies were reduced following cycloheximide treatment (which disrupts P-bodies) and were increased following puromycin treatment (which induces P-body formation) (data not shown). These observations made me confident that I have the tools to determine whether T and B lymphocytes contain P-bodies.  4.2. Characterization of P-bodies in T lymphocytes 4.2.1. T lymphocytes contain GWBs and express the P-body markers GW182, RCK/p54, and DCP1a To assess whether T lymphocytes contain P-bodies, I have analyzed expression of GWBs and the P-body markers GW182, RCK/p54, and DCP1a by western immunoblotting and flow cytometry in addition to assessing their intracellular  76  localization by confocal microscopy. For this analysis, primary murine CD4+ and CD8+ T lymphocytes were isolated from the spleen and lymph nodes of wild-type (WT) B6 mice with high purity (Figure 4.1). It is important to note that primary murine T-cell populations contain a subset on memory-phenotype (MP) CD4+ and CD8+ T lymphocytes (CD3+ CD4+ CD44High and CD3+ CD8+ CD44High, respectively; Figure 4.1), and thus are not identical to naïve T lymphocytes (Chapter 5). Immunoblotting analysis of Jurkat (human CD4+), MOLT4 (human CD4+), HBPALL (human CD8+), DO11.10 (murine CD4+), EL4 (murine CD4+), and CTLL2 (murine CD8+) T-cell lines, as well as primary CD4+ and CD8+ T lymphocytes from WT mice, showed that T lymphocytes express GW182, RCK/p54, and DCP1a. The human antiGWB serum 18033 recognized several bands that are likely to be Ge-1, RAP55, and DCP2 (data from Kate Choi; Figure 4.2). In it’s original description, the ~180 kDa band recognized by this anti-GWB serum was thought to be GW182 112. However, it was later determined that this band is in fact another P-body marker, Ge-1 185. Furthermore, I detected GWBs, GW182, RCK/p54, and DCP1a in Jurkat (human CD4+), DO11.10 (murine CD4+), HBP-ALL (human CD8+), and CTLL2 (murine CD8+) T-cell lines, as well as in primary murine CD4+ and CD8+ T lymphocytes (Figure 4.3A, Figure 4.5A, and data not shown) through dual analysis by flow cytometry and confocal microscopy (Illustrations 4.1 and 4.2). In addition, I found that MOLT4 (human CD4+) and EL4 (murine CD4+) T-cell lines express GWBs, GW182, RCK/p54, and DCP1a via flow cytometry (data not shown). Overall, western immunoblotting and flow cytometry data indicates that T lymphocytes express P-body markers.  77  Figure 4.1. Primary CD4+ and CD8+ T lymphocytes were isolated to high purity from WT mice and contain a subset of memory-phenotype cells. Primary CD4+ and CD8+ T lymphocytes were isolated from spleens and lymph nodes of B6 mice via negative selection and analyzed by flow cytometry. This technique allows isolation of highly pure CD4+ or CD8+ T cells (CD3+ B220-). Even though these T cells were isolated from naïve mice, there is a prominent memory-phenotype population (CD44High) of 10-13%. Results are representative of >5 independent experiments.  78  Figure 4.2. CD4+ and CD8+ T lymphocytes express GW182, RCK/p54, DCP1a, and the P-body markers detected by the reference anti-GWB serum. 40 µg of lysates from HEK 293 cells (positive control), Jurkat (human CD4+), MOLT4 (human CD4+), HBP-ALL (human CD8+), DO11.10 (murine CD4+), EL4 (murine CD4+), and CTLL2 (murine CD8+) Tcell lines, as well as primary CD4+ and CD8+ T lymphocytes from spleens and lymph nodes of B6 mice were analyzed by western immunoblotting for expression of GWBs, GW182, RCK/p54, and DCP1a. Blots were re-probed with Abs against actin and α-tubulin as loading controls, and re-probes from one blot are shown (GW182). Results are representative of 3 independent experiments.  79  4.2.2. GWBs, GW182, RCK/p54, and DCP1a are localized to discrete cytoplasmic foci in CD4+ T lymphocytes In addition to expression of P-body markers, dual analysis by flow cytometry and confocal microscopy (Illustration 4.1), showed that in Jurkat (human CD4+) and DO11.10 (murine CD4+) T-cell lines, as well as in primary murine CD4+ T lymphocytes, GWBs, GW182, RCK/p54, and DCP1a are localized to discrete cytoplasmic foci (Figure 4.3B and data not shown). Surprisingly, the number of granules and the cumulative granule area per cell was different for each of the P-body markers that were analyzed (Figure 4.3C and Figure 4.3D). This suggests that there are different subsets of P-bodies that contain different P-body markers and which could potentially have different functions. For each P-body marker, the granules containing that marker exhibited a wide range of size, although the majority of all granules had a diameter ≤ 0.6 µm (Figure 4.3E). Interestingly, most of the GW182 granules were ≤ 0.3 µm and the greatest proportion of DCP1a granules were 0.3-0.4 µm. The majority of GWBs and RCK/p54 granules were almost equally distributed between the ≤ 0.3 µm and 0.3-0.4 µm groups. In summary, I have shown that P-body markers are expressed in CD4+ T lymphocytes and are concentrated in discrete cytoplasmic granules. Therefore, CD4+ T lymphocytes contain Pbodies. 4.2.3. CD4+ T lymphocytes contain different P-body subsets Based on my initial observations that different P-body markers are present in Pbody-like granules to different extents, it was important to determine whether there are subsets of P-bodies with different protein compositions. Therefore, I assessed the colocalization of GWBs, GW182, RCK/p54, and DCP1a by confocal microscopy,  80  Illustration 4.1. Method outline for dual analysis of P-body markers in primary murine CD4+ T lymphocytes by flow cytometry and confocal microscopy. Primary CD4+ T lymphocytes from spleens and lymph nodes of B6 mice were fixed in 4% PFA, permeabilized with SWB, incubated with blocking buffer, and stained in suspension with 1º Abs specific for GWBs, GW182, RCK/p54, or DCP1a and 2º Abs conjugated to AlexaFluor488 (green) in combination with rhodamine-phalloidin (red) to mark F-actin. ~25% of the cells were injected into the mounting medium with DAPI (blue) to visualize by confocal microscopy and the remaining ~75% of the cells were analyzed by flow cytometry.  81  Figure 4.3. Primary murine CD4+ T lymphocytes contain GWBs and express GW182, RCK/p54, and DCP1a that are localized to discrete cytoplasmic foci.  82  Primary CD4+ T lymphocytes from spleens and lymph nodes of B6 mice were stained in suspension with 1º Abs specific for GWBs, GW182, RCK/p54, or DCP1a and 2º Abs conjugated to Alexa Fluor 488 (green). Normal human serum, mouse IgG1, and rabbit IgG at matched concentrations were used for isotype control staining of GWBs, GW182, and RCK/p54 and DCP1a, respectively. These cells were also stained with rhodamine-phalloidin (red) to mark F-actin. (A) ~75% of the cells were analyzed by flow cytometry to analyze expression levels of P-body markers and demonstrate consistency of staining on a population level. Note, F-actin staining demonstrates that the samples received the same amount of the 2º Ab master mix. (B) ~25% of the cells were injected into mounting medium with DAPI (blue) and visualized by confocal microscopy. CD4+ T lymphocytes contain each P-body marker localized to granules (green) within the cytoplasm between nucleus (blue) and cortical F-actin (red). Results in (A) and (B) are representative of 34 independent experiments. (C-E) For each P-body marker, the number of granules (C), cumulative granule area per cell (D), and granule size distribution (E) were quantified based on >120 cells from 3 independent experiments. Mean ± standard deviation (SD). *p < 0.05, **p < 0.001, ***p < 0.0001.  83  determined Pearson’s correlation coefficient, and determined M1 and M2 co-occurrence coefficients for all the possible combinations of P-body markers (RCK/p54 and DCP1a could not be co-stained, because the available Abs are both rabbit Abs). In fluorescent microscopy, Pearson’s correlation coefficient measures the degree of linear dependence (statistical correlation) between two fluorophores and can have values between -1 (inverse correlation) and 1 (complete correlation) inclusive. M1 and M2 co-occurrence coefficients assess the degree of spatial overlap (co-occurrence in the same pixel) between two fluorophores and can have values between 0 (no overlap) and 1 (complete overlap) inclusive. M1 coefficient measures occurrence of fluorophore 2 pixels in pixels containing fluorophore 1, and vice versa M2 coefficient measures occurrence of fluorophore 1 pixels in pixels containing fluorophore 2. In primary CD4+ T lymphocytes from WT mice, GWBs were highly co-localized with RCK/p54 granules and partially co-localized with DCP1a granules (Pearson’s correlation coefficients 0.72 ± 0.10 and 0.46 ± 0.12, respectively; Figure 4.4A and Figure 4.4C). However, GW182 foci were rarely co-localized with the other P-body markers (Pearson’s correlation coefficients for GW182 vs. GWBs, RCK/p54, and DCP1a < 0.08). Co-occurrence analysis revealed that most of the RCK/p54 and DCP1a granules were also positive for GWB staining (M1 overlap coefficients 0.83 ± 0.12 and 0.74 ± 0.12, respectively; Figure 4.4D), even though only ~70-80% of GWBs contained RCK/p54 and only ~30-40% of GWBs contained DCP1a (M2 overlap coefficients 0.65 ± 0.08 and 0.29 ± 0.09, respectively). In contrast, GW182 marked an entirely different subset of P-bodies (M1 and M2 overlap coefficients for GW182 vs. GWBs, RCK/p54, and DCP1a < 0.18). P-body marker distribution in a representative primary murine CD4+ T lymphocyte is  84  Figure 4.4. Primary murine CD4+ T lymphocytes contain subsets of P-bodies with various compositions of GWBs, GW182, RCK/p54, and DCP1a. Primary CD4+ T lymphocytes from spleens and lymph nodes of B6 mice were stained in suspension with 1º Abs specific for two of GWBs, GW182, RCK/p54, or DCP1a and 2º Abs conjugated to either Alexa Fluor 488 (green) or Alexa Fluor 647 (red). The cells were subsequently injected into mounting medium with  85  DAPI (blue) and visualized by confocal microscopy (A). Results in (A) are representative of 3-4 independent experiments. (B) Diagram of a representative primary murine CD4+ T lymphocyte containing distinct subsets of P-bodies with different compositions of GWBs, GW182, RCK/p54, or DCP1a. (C-D) Co-localization (Pearson’s correlation coefficient, C) and co-occurrence (M1 and M2 overlap coefficients, D) of P-body markers were quantified based on >120 cells from 3 independent experiments. Mean ± SD. *p < 0.05, **p < 0.001, ***p < 0.0001.  86  shown in Figure 4.4B. These results were supported by similar observations in the Jurkat (human CD4+) and DO11.10 (murine CD4+) T-cell lines (data not shown). Therefore, in CD4+ T lymphocytes there exist subsets of P-bodies with different protein compositions and possibly distinct functional properties, such as translational repression or mRNA degradation. Recent evidence suggests that GW182 and DCP1a also accumulate in distinct granules in cultured monocytes 186, supporting this observation. However, P-body protein composition seems to be cell type-specific, because GW182 and DCP1a partially colocalize in HEp-2 cells 112, 115. 4.2.4. GWBs, GW182, RCK/p54, and DCP1a are localized to discrete cytoplasmic foci in CD8+ T lymphocytes Using dual analysis by flow cytometry and confocal microscopy (Illustration 4.2), I also found that GWBs, GW182, RCK/p54, and DCP1a were localized in discrete cytoplasmic foci in HBP-ALL (human CD8+) and CTLL2 (murine CD8+) T-cell lines, as well as in primary murine CD8+ T lymphocytes (Figure 4.5B and data not shown). The number of granules and the cumulative area of granules per cell were different for most of the P-body marker pairs, with similarities observed for GWBs and RCK/p54, and for GW182 and DCP1a (Figure 4.5C and Figure 4.5D). Therefore, distinct subsets of Pbodies appear to exist CD8+ T cells. As with CD4+ T cells, the size of the granules was variable for each P-body marker analyzed, but the majority of granules had a diameter ≤ 0.6 µm for all P-body markers (Figure 4.5E). Similar to CD4+ T cells, most of the GW182 granules had a diameter ≤ 0.3 µm whereas most DCP1a granules were 0.3-0.4 µm. Most of GWBs and RCK/p54 granules were almost equally distributed between the  87  Illustration 4.2. Method outline for dual analysis of P-body markers in primary murine CD8+ T lymphocytes by flow cytometry and confocal microscopy. Primary CD8+ T lymphocytes from spleens and lymph nodes of B6 mice were fixed in 4% PFA, permeabilized with SWB, incubated with blocking buffer, and stained in suspension with 1º Abs specific for GWBs, GW182, RCK/p54, or DCP1a and 2º Abs conjugated to AlexaFluor488 (green) in combination with rhodamine-phalloidin (red) to mark F-actin. ~25% of the cells were injected into the mounting medium with DAPI (blue) to visualize by confocal microscopy and the remaining ~75% of the cells were analyzed by flow cytometry.  88  Figure 4.5. Primary murine CD8+ T lymphocytes contain GWBs and express GW182, RCK/p54, and DCP1a that are localized to discrete cytoplasmic foci.  89  Primary CD8+ T lymphocytes from spleens and lymph nodes of B6 mice were stained in suspension with 1º Abs specific for GWBs, GW182, RCK/p54, or DCP1a and 2º Abs conjugated to Alexa Fluor 488 (green). Normal human serum, mouse IgG1, and rabbit IgG at matched concentrations were used for isotype control staining of GWBs, GW182, and RCK/p54 and DCP1a, respectively. These cells were also stained with rhodamine-phalloidin (red) to mark F-actin. (A) ~75% of the cells were analyzed by flow cytometry to analyze expression levels of P-body markers and demonstrate consistency of staining on a population level. Note, F-actin staining demonstrates that the samples received the same amount of the 2º Ab master mix. (B) ~25% of the cells were injected into mounting medium with DAPI (blue) and visualized by confocal microscopy. CD8+ T lymphocytes contain each P-body marker localized to granules (green) within the cytoplasm between nucleus (blue) and cortical F-actin (red). Results in (A) and (B) are representative of 34 independent experiments. (C-E) For each P-body marker, the number of granules (C), cumulative granule area per cell (D), and granule size distribution (E) were quantified based on >120 cells from 3 independent experiments. Mean ± SD. *p < 0.05, **p < 0.001, ***p < 0.0001.  90  ≤ 0.3 µm and 0.3-0.4 µm groups. Overall, I have shown that P-body markers are expressed in CD4+ and CD8+ T lymphocytes and concentrate in discrete cytoplasmic granules. Thus, T lymphocytes contain granules that can be classified as P-bodies. 4.2.5. CD8+ T lymphocytes contain different P-body subsets Co-localization analysis of GWBs, GW182, RCK/p54, and DCP1a by confocal microscopy in primary murine CD8+ T lymphocytes confirmed that GWBs were highly co-localized with RCK/p54 granules and partially co-localized with DCP1a granules (Pearson’s correlation coefficients 0.75 ± 0.05 and 0.38 ± 0.09, respectively; Figure 4.6A and Figure 4.6C), whereas GW182 foci were infrequently co-localized with the other Pbody markers (Pearson’s correlation coefficients for GW182 vs. GWBs, RCK/p54, and DCP1a < 0.07). As in CD4+ T cells, most of the RCK/p54 and DCP1a granules were also positive for GWB staining (M1 overlap coefficients 0.95 ± 0.04 and 0.81 ± 0.08, respectively; Figure 4.6D), even though only ~70-80% of GWBs contained RCK/p54 and only ~40-50% of GWBs had DCP1a (M2 overlap coefficients 0.62 ± 0.09 and 0.23 ± 0.08, respectively). GW182 marked a distinct subset of P-bodies (M1 and M2 overlap coefficients for GW182 vs. GWBs, RCK/p54, and DCP1a < 0.14). P-body marker distribution in a representative primary murine CD8+ T lymphocyte is shown in Figure 4.6B. These results were supported by similar findings in HBP-ALL (human CD8+) and CTLL2 (murine CD8+) T-cell lines (data not shown). Therefore, similar P-body subsets exist in both CD4+ and CD8+ T lymphocytes.  91  Figure 4.6. Primary murine CD8+ T lymphocytes contain subsets of P-bodies with various compositions of GWBs, GW182, RCK/p54, and DCP1a. Primary CD8+ T lymphocytes from spleens and lymph nodes of B6 mice were stained in suspension with 1º Abs specific for two of GWBs, GW182, RCK/p54, or DCP1a and 2º Abs conjugated to either Alexa Fluor 488 (green) or Alexa Fluor 647 (red). The cells were subsequently injected into mounting medium with  92  DAPI (blue) and visualized by confocal microscopy (A). Results in (A) are representative of 3-4 independent experiments. (B) Diagram of a representative primary murine CD8+ T lymphocyte containing distinct subsets of P-bodies with different compositions of GWBs, GW182, RCK/p54, or DCP1a. (C-D) Co-localization (Pearson’s correlation coefficient, C) and co-occurrence (M1 and M2 overlap coefficients, D) of P-body markers were quantified based on >120 cells from 3 independent experiments. Mean ± SD. *p < 0.05, **p < 0.001, ***p < 0.0001.  93  4.3. Characterization of P-bodies in B lymphocytes 4.3.1. B lymphocytes contain GWBs and express P-body markers GW182, RCK/p54, and DCP1a To determine whether B lymphocytes contain P-bodies, I analyzed the expression of GWBs and the P-body markers GW182, RCK/p54, and DCP1a by immunoblotting and flow cytometry, and also assessed their intracellular localization by confocal microscopy. For this analysis, primary murine B lymphocytes were isolated from the spleens of WT B6 mice with high purity (Figure 4.7). Immunoblotting analysis of RAMOS (human IgM+), BJAB (human IgM+), OPM2 (human myeloma), K40B-1 (murine pro-B cell-like, IgM-), K40B-2 (murine pre-B celllike, IgM+), WEHI-231 (murine immature, IgM+), A20 (murine mature, IgG+), 5TGM1 (murine myeloma), MPC-11 (murine myeloma, IgG-secreting) B-cell lines, as well as primary B lymphocytes from WT mice, showed that B lymphocytes express GW182, RCK/p54, and DCP1a. The human anti-GWB reference serum 18033 recognized the same bands as in T cells that I identified as Ge-1, RAP55, and DCP2 (data from Kate Choi; Figure 4.8). The observation that the anti-GW182 mAb recognized a ~220-260 kDa band, whereas the anti-GWB serum reacts with a ~180 kDa band confirms the previous report that the ~180 kDa band is not GW182 and is in fact Ge-1 185. Moreover, I have confirmed the detection of GWBs, GW182, RCK/p54, and DCP1a in RAMOS (human IgM+) and WEHI-231 (murine IgM+) B-cell lines, as well as in primary murine B lymphocytes (Figure 4.9A and data not shown) through dual analysis by flow cytometry and confocal microscopy (Illustrations 4.3). Together, the immunoblotting and flow cytometry data indicate that both T and B lymphocytes express P-body markers. 94  Figure 4.7. Primary B lymphocytes were isolated to high purity from WT mice. Primary B lymphocytes were isolated from spleens of B6 mice via negative selection and analyzed by flow cytometry. This technique allows isolation of highly pure B cells (B220+ CD3-). These B cells were isolated from naïve mice and display naïve phenotype (IgM+). Results are representative of >5 independent experiments.  95  Figure 4.8. B lymphocytes express GW182, RCK/p54, DCP1a, and the P-body markers detected by the reference anti-GWB serum. 40 µg of lysates from HEK 293 cells (positive control), RAMOS (human immature, IgM+), BJAB (human IgM+), OPM2 (human myeloma), K40B-1 (murine pro-B cell-like, IgM-), K40B-2 (murine pre-B cell-like, IgM+), WEHI-231 (murine immature, IgM+), A20 (murine mature, IgG+), 5TGM1 (murine myeloma), MPC-11 (murine myeloma, IgG-secreting) B-cell lines, as well as primary B lymphocytes from spleens of B6 mice were analyzed by western immunoblotting for expression of GWBs, GW182, RCK/p54, and DCP1a. Blots were re-probed with Abs against actin and α-tubulin as loading controls, and re-probes from one blot are shown (GW182). Results are representative of 3 independent experiments.  96  4.3.2. GWBs, GW182, RCK/p54, and DCP1a are localized to discrete cytoplasmic foci in B lymphocytes Using dual analysis by flow cytometry and confocal microscopy (Illustration 4.3), I found that GWBs, GW182, RCK/p54, and DCP1a are localized to discrete cytoplasmic foci in RAMOS (human IgM+) and WEHI-231 (murine IgM+) B-cell lines, as well as in primary murine B lymphocytes (Figure 4.9B and data not shown). The cumulative area of granules per cell was different for all the P-body marker pairs, although the number of granules per cell was only different for GW182 (Figure 4.9C and Figure 4.9D). Therefore, distinct subsets of P-bodies could exist in B cells. The size of the granules in primary murine B lymphocytes was even more variable than in T cells for each P-body marker analyzed. The diameter of GWBs, RCK/p54, and DCP1a granules ranged from ≤ 0.3 µm to > 0.7 µm, but the majority of GW182 granules was ≤ 0.4 µm (Figure 4.9E). To conclude, I have shown that P-body markers are expressed in T and B lymphocytes and are concentrated in discrete cytoplasmic granules. 4.3.2. B lymphocytes contain two subsets of P-bodies Confocal microscopy co-localization analysis of GWBs, GW182, RCK/p54, and DCP1a in primary murine B lymphocytes showed that GWBs were highly co-localized with both RCK/p54 and DCP1a granules (Pearson’s correlation coefficients 0.83 ± 0.08 and 0.80 ± 0.06, respectively; Figure 4.10A and Figure 4.10C), whereas GW182 foci were seldom co-localized with the other P-body markers (Pearson’s correlation coefficients for GW182 vs. GWBs, RCK/p54, and DCP1a < 0.06). As in T cells, most of the RCK/p54 and DCP1a granules were also positive for GWB staining (M1 overlap coefficients 0.95 ± 0.05 and 0.90 ± 0.05, respectively; Figure 4.10D). However, most of  97  Illustration 4.3. Method outline for dual analysis of P-body markers in primary murine B lymphocytes by flow cytometry and confocal microscopy. Primary B lymphocytes from spleens of B6 mice were fixed in 4% PFA, permeabilized with SWB, incubated with blocking buffer, and stained in suspension with 1º Abs specific for GWBs, GW182, RCK/p54, or DCP1a and 2º Abs conjugated to AlexaFluor488 (green) in combination with rhodaminephalloidin (red) to mark F-actin. ~25% of the cells were injected into the mounting medium with DAPI (blue) to visualize by confocal microscopy and the remaining ~75% of the cells were analyzed by flow cytometry.  98  Figure 4.9. Primary murine B lymphocytes contain GWBs and express GW182, RCK/p54, and DCP1a that are localized to discrete cytoplasmic foci. 99  Primary B lymphocytes from spleens of B6 mice were stained in suspension with 1º Abs specific for GWBs, GW182, RCK/p54, or DCP1a and 2º Abs conjugated to Alexa Fluor 488 (green). Normal human serum, mouse IgG1, and rabbit IgG at matched concentrations were used for isotype control staining of GWBs, GW182, and RCK/p54 and DCP1a, respectively. These cells were also stained with rhodaminephalloidin (red) to mark F-actin. (A) ~75% of the cells were analyzed by flow cytometry to analyze expression levels of P-body markers and demonstrate consistency of staining on a population level. Note, F-actin staining demonstrates that the samples received the same amount of the 2º Ab master mix. (B) ~25% of the cells were injected into mounting medium with DAPI (blue) and visualized by confocal microscopy. B lymphocytes contain each P-body marker localized to granules (green) within the cytoplasm between nucleus (blue) and cortical F-actin (red). Results in (A) and (B) are representative of 3-4 independent experiments. (C-E) For each P-body marker, the number of granules (C), cumulative granule area per cell (D), and granule size distribution (E) were quantified based on >120 cells from 3 independent experiments. Mean ± SD. *p < 0.05, **p < 0.001, ***p < 0.0001.  100  Figure 4.10. Primary murine B lymphocytes contain two subsets of P-bodies: GWB+ RCK/p54+ DCP1a+ and GW182+. Primary B lymphocytes from spleens of B6 mice were stained in suspension with 1º Abs specific for two of GWBs, GW182, RCK/p54, or DCP1a and 2º Abs conjugated to either Alexa Fluor 488 (green) or Alexa  101  Fluor 647 (red). The cells were subsequently injected into mounting medium with DAPI (blue) and visualized by confocal microscopy (A). Results in (A) are representative of 3-4 independent experiments. (B) Diagram of a representative primary murine B lymphocyte containing distinct subsets of P-bodies with different compositions of GWBs, GW182, RCK/p54, or DCP1a. (C-D) Co-localization (Pearson’s correlation coefficient, C) and co-occurrence (M1 and M2 overlap coefficients, D) of P-body markers were quantified based on >120 cells from 3 independent experiments. Mean ± SD. *p < 0.05, **p < 0.001, ***p < 0.0001.  102  RCK/p54 and DCP1a granules also contained GWBs (M2 overlap coefficients 0.75 ± 0.10 and 0.67 ± 0.14, respectively). In contrast, GW182 was localized to a distinct subset of P-bodies (M1 and M2 overlap coefficients for GW182 vs. GWBs, RCK/p54, and DCP1a < 0.17). P-body marker distribution in a representative primary murine B lymphocyte is shown in Figure 4.10B. These results were supported by similar observations in RAMOS (human IgM+) and WEHI-231 (murine IgM+) B-cell lines (data not shown). Therefore, based on the markers analyzed, there are two main P-body subsets in B lymphocytes: GWB+ RCK/p54+ DCP1a+ granules and GW182+ granules. This raises the possibility that P-bodies, with different protein compositions and potentially distinct functional properties, exist in B cells.  4.4. Differences in P-body subsets between T and B lymphocytes Upon direct comparison of primary CD4+ T, CD8+ T, and B lymphocytes from WT mice, it became apparent that P-bodies in CD4+ and CD8+ T lymphocytes are quite similar. In contrast, there were a few notable differences between P-bodies in T and B cells. First, B cells contained somewhat fewer GWB+ and RCK/p54+ P-bodies, but slightly more DCP1a+ P-bodies (Figure 4.11A). Second, DCP1a+ P-bodies were somewhat larger in B cells than in T cells (Figure 4.11B). Third, B cells contained somewhat fewer small (≤ 0.4 µm) and a correspondingly greater proportion of large (> 0.5 µm) GWB+, RCK/p54+, and DCP1a+ P-bodies (Figure 4.11C). Finally, even though GWBs were partially co-localized with DCP1a in T cells, nearly all GWBs contained DCP1a in B cells (Figure 4.11D and Figure 4.11E). However, there were no notable differences in GW182+ P-bodies between T and B cells. In conclusion, (1) primary B lymphocytes contain somewhat fewer P-bodies than primary T lymphocytes, but some of 103  Figure 4.11. Differences in P-body subsets between T and B lymphocytes.  104  Primary CD4+ and CD8+ T lymphocytes or B lymphocytes were isolated simultaneously from spleens and lymph nodes or spleens of B6 mice, respectively. All cells were stained in suspension with 1º Abs specific for GWBs, GW182, RCK/p54, or DCP1a and 2º Abs conjugated to either Alexa Fluor 488 or Alexa Fluor 647. The cells were subsequently injected into mounting medium with DAPI and visualized by confocal microscopy. (A-E) For each P-body marker, the number of granules (A), cumulative granule area per cell (B), and granule size distribution (C), as well as co-localization (Pearson’s correlation coefficient, D) and co-occurrence (M1 and M2 overlap coefficients, related groups are encircled, E) of P-body markers, were quantified based on >80 cells from 2 independent experiments where these cells were analyzed simultaneously. Mean ± SD. *p < 0.05, **p < 0.001, ***p < 0.0001.  105  the B-cell P-bodies were larger than those in T cells; (2) there seems to be three major Pbody subsets in T cells: GWB+ RCK/p54+ DCP1a+ P-bodies, GWB+ RCK/p54+ P-bodies, and GW182+ P-bodies, whereas B cells contain two major P-body subsets: GWB+ RCK/p54+ DCP1a+ P-bodies and GW182+ P-bodies.  4.5. Analysis of P-bodies in B lymphocytes following activation To assess changes in the number, composition, or localization P-bodies following B-cell activation, I stimulated primary B lymphocytes from WT mice with (1) αIgM Abs, (2) a combination of αIgM and αCD40 Abs plus IL-4, or (3) αCD40 Abs plus IL-4 for 20 minutes, 1 hour, 6 hours, or 24 hours (Illustration 4.4). These treatments successfully activated the B cells, as indicated by downregulation of their surface and intracellular IgM (only in response to αIgM stimulation), expression of CD86, and increased F-actin content (Figure 4.12A). Remarkably, flow cytometry showed that the levels of GWBs, GW182, and DCP1a (but not RCK/p54) were downregulated immediately (at 20 minutes, 1 hour, and 6 hours) following activation, even though statistical significance was only achieved at 1 hour for GWBs and DCP1a. The remaining P-bodies seemed to be dissociated and were localized in proximity of clustered IgM at 1 hour (and somewhat at 20 minutes and 6 hours) following activation if BCR was engaged (Figures 4.12C-F and data not shown). Strikingly, DCP1a granules were either completely or almost completely dissociated (Figure 4.12F). In contrast, after 24 hours, all P-body markers were upregulated in primary murine B lymphocytes that had been exposed to αIgM Abs, which cluster their BCRs (Figure 4.12B). Moreover, primary murine B lymphocytes that had been activated  106  Illustration 4.4. Method outline for analysis on P-bodies in primary murine B lymphocytes activated with αIgM and/or αCD40 and IL-4. Primary B lymphocytes from spleens of B6 mice were activated with αIgM, IL-4 + αCD40 + αIgM, or IL-4 + αCD40 for 20 minutes, 1 hour, 6 hours, or 24 hours, fixed in 4% PFA, permeabilized with SWB, incubated with blocking buffer, and stained in suspension with 1º Abs specific for GWBs, GW182, RCK/p54, or DCP1a and 2º Abs conjugated to Alexa Fluor 488 (green) in combination with rhodaminephalloidin (red) to mark F-actin and αIgM-Alexa Fluor 647 to detect clustered IgM (violet). ~25% of the cells were injected into the mounting medium with DAPI (blue) to visualize by confocal microscopy and the remaining ~75% of the cells were analyzed by flow cytometry.  107  108  109  Figure 4.12. Changes in P-bodies of primary murine B lymphocytes following activation with αIgM and/or αCD40 and IL-4. Primary B lymphocytes from spleens of B6 mice were activated with αIgM, IL-4 + αCD40 + αIgM, or IL-4 + αCD40 for 20 minutes, 1 hour, 6 hours, or 24 hours and fixed in 4% PFA. (A) Some of the cells were analyzed for expression of surface IgM, CD86, and B220 by flow cytometry. (B-F) Remaining cells were stained in suspension with 1º Abs specific for GWBs, GW182, RCK/p54, or DCP1a and 2º Abs conjugated to Alexa Fluor 488 (green), rhodamine-phalloidin (red) to mark F-actin, and αIgM-Alexa Fluor 647 to detect clustered IgM (violet). (B) ~75% of the cells were analyzed by flow cytometry to determine  110  expression levels of P-body markers, total IgM, and F-actin as geometric mean fluorescent intensity (MFI) relative to unstimulated cells. Mean ± SEM. Results are based on 2 independent experiments. (C-F) ~25% of the cells were injected into mounting medium with DAPI (blue) and visualized by confocal microscopy. Results in (A) and (C-F) are representative of 2-3 independent experiments. Unless indicated, asterisks show significance relative to unstimulated cells. *p < 0.05, **p < 0.001, ***p < 0.0001.  111  with αIgM Abs for 24 hours also seemed to contain increased number of granules detected by each P-body marker (Figures 4.12C-F and data not shown). I have confirmed some of these findings in Jurkat (human CD4+) T cell-line and in primary murine CD8+ T lymphocytes by activating these cells with beads coated with αCD3 and αCD28 Abs (data not shown). However, the rate of bead-cell conjugate formation was low, making the data difficult to interpret. Therefore, I have assessed changes in P-bodies of T lymphocytes following immune synapse formation with Agbearing DCs in Chapter 5. In conclusion, activation of primary B lymphocytes leads to downregulation of GWBs, GW182, and DCP1a, but not RCK/p54, as well as to partial dissociation of Pbodies. In αIgM-stimulated cells, the remaining P-bodies seem to accumulate in proximity of the clustered IgM at 1 hour. Finally, the levels of all P-body markers are upregulated at 24 hours if BCR was engaged.  4.6. Discussion 4.6.1. Analysis of P-bodies in T and B lymphocytes In this Chapter, I addressed hypothesis 1 and established that: Conclusion 1: T and B lymphocytes contain P-bodies. Specifically, via immunoblotting and dual analysis by flow cytometry and confocal microscopy, I found that human and murine T and B-cell lines, as well as primary murine CD4+ T, CD8+ T, and B lymphocytes contain GWBs and express the Pbody markers GW182, RCK/p54, and DCP1a, and that these P-body markers are 112  concentrated in discrete cytoplasmic granules. Moreover, I confirmed that in T and B lymphocytes, as previously published for HEp-2 cells 185, the ~180 kDa band detected by the reference human anti-GWB serum 18033 is not GW182, and therefore is likely to be another P-body marker, Ge-1, as found by the authors of Ref. 185. Furthermore, based on the markers analyzed, I determined that: Conclusion 2: There are three main P-body subsets in T lymphocytes: GWB+ RCK/p54+, GWB+ RCK/p54+ DCP1a+, and GW182+ P-bodies. Conclusion 3: There are two main P-body subsets in B lymphocytes: GWB+ RCK/p54+ DCP1a+ and GW182+ P-bodies. Explicitly, using confocal microscopy, I measured the number of P-bodies per cell, size distribution of P-bodies, and cumulative area per cell for each P-body marker in human and murine T and B-cell lines, as well as primary murine CD4+ T, CD8+ T, and B lymphocytes. In addition, I quantified the co-localization and co-occurrence of all possible P-body marker pairs. I found that P-bodies are heterogeneous in size and protein composition, but that the number of P-bodies and the cumulative area per cell for each Pbody marker is fairly constant, at least for primary cells. Nevertheless, it is important to note that due to the resolution limits of confocal microscopy (x-y and z resolution is ~200 nm and ~500 nm, respectively), I was not able to distinguish one large P-body from several smaller ones that are closer than these resolution limits. Therefore, it is possible that I could have underestimated the number of P-bodies in the cells analyzed. Furthermore, I established that GWBs co-localize with RCK/p54 granules, whereas GW182 marks a separate subset of P-bodies in both T and B lymphocytes. 113  However, I found that DCP1a is found in most of GWBs in B lymphocytes, but only in approximately half of the GWBs in T lymphocytes, whereas DCP1a is rarely separate from GWBs. Based on these results, I determined that three main P-body subsets exist in T lymphocytes: GWB+ RCK/p54+ DCP1a+, GWB+ RCK/p54+, and GW182+ P-bodies, and that there are only two main P-body subsets in B lymphocytes: GWB+ RCK/p54+ DCP1a+ and GW182+ P-bodies. This raises the possibility that there are P-bodies with different protein compositions, which could have distinct functional properties, such as translational repression or mRNA degradation. The observation that DCP1a does not co-localize with GW182 in either T or B cells is in agreement with the recent evidence that GW182 and DCP1a are also found in distinct granules in cultured monocytes 186. Nevertheless, P-body protein composition could be cell type-specific, because GW182 and DCP1a at least partially co-localize in HEp-2 cells 112, 115 and in HEK 293 cells 184, and there are discrepancies in P-body subsets even between T and B lymphocytes. GW182 was consistently reported to co-localize with the miRNA effector protein Argonaute 2 (AGO2) 184, 186. GW182 and AGO2, two main components of the RNAinduced silencing complex (RISC) 116-118, 187, are part of the miRNA-loaded RISC (miRISC) and are associated with endosomes, endolysosomal compartments called multivesicular bodies (MVBs), and secreted vesicles called exosomes (reviewed in Ref. 188  ), which derive from MVBs 186. Furthermore, I found that the majority of GW182+ P-  bodies are ≤ 0.3 µm and have the smallest average diameter. Therefore, it is conceivable that GW182+ P-bodies are aggregates of miRISCs that are waiting to be sorted for storage, degradation, or secretion, with GW182 possibly recycled if the target mRNA’s 114  destination is another subset of P-bodies. Considering the role of DCP1a in mRNA decapping 119, 120, DCP1a+ P-bodies could be considered degradative. Since RCK/p54 is a general translation repressor protein that is required for some miRNAs to function, and is downregulated under certain conditions to allow the recruitment of translationallyrepressed mRNAs back into the translationally-active pool 109, 118, RCK/p54+ P-bodies might be storing miRISCs that have been sorted for storage by GW182. Consequently, one could speculate that GW182+ P-bodies are aggregates of miRISCs that are subsequently sorted for storage into GWB+ RCK/p54+ P-bodies, degradation in GWB+ RCK/p54+ DCP1a+ P-bodies, or secretion (e.g. via exosomes). Moreover, the decay promoting/destabilizing ARE-BP TTP can deliver AREcontaining mRNAs into RCK/p54+ DCP1a+ P-bodies 183, but stabilizing ARE-BPs, such as HuR, exist 150-152 (reviewed in Refs. 134, 153, 154). Therefore, the decay promoting/destabilizing ARE-BPs could deliver specific translationally-repressed mRNAs into GWB+ RCK/p54+ DCP1a+ P-bodies for degradation, whereas stabilizing ARE-BPs could potentially transport other translationally-repressed mRNAs into GWB+ RCK/p54+ P-bodies for storage. Consequently, both GWB+ RCK/p54+ P-bodies and GWB+ RCK/p54+ DCP1a+ P-bodies potentially contain mRNAs delivered in a miRNAindependent manner. It is an important future direction to determine the specific roles of P-body subsets in T and B lymphocytes. 4.6.2. Evaluation of P-body localization to the model immune synapse in B lymphocytes In this Chapter, I also tested hypothesis 3 in B lymphocytes and found that: Conclusion 4: In B lymphocytes, P-bodies partially dissociate in response to BCR cross115  linking, and the remaining GWB+ RCK/p54+ DCP1a+ P-bodies localize in proximity of the model immune synapse, whereas remaining GW182+ P-bodies localize at the model immune synapse. Specifically, I found that in primary murine B lymphocytes, cross-linking of BCRs in response to stimulation with αIgM Abs, either alone or in the presence of αCD40 Abs and IL-4, induces GWBs, RCK/p54, and DCP1a to partially dissociate and to localize in proximity of the model immune synapse (clustered BCRs), whereas GW182 localizes at the model immune synapse. In other words, partial dissociation can be described as the sum of observations that P-bodies become less defined as discrete foci (become fainter and in some cases fragmented), and that diffuse P-body staining can be observed in the cytoplasm in the vicinity of remaining P-bodies. This re-localization of Pbodies towards the model immune synapse, as well as their partial dissociation, is intriguing. Even though I was not able to test whether specific mRNAs are released for translation following movement of P-bodies towards the model immune synapse, it is possible that the mobilization of mRNAs from P-bodies plays an important role in B-cell activation and/or cell fate specification. It is important to note, however, that the data presented here is limited to the analysis of the model immune synapse formed by crosslinked BCRs, and the detection of P-bodies relative to the immune synapse formed by B lymphocytes and APCs would strengthen conclusion 4. Additionally, I found that GWBs, GW182, and DCP1a, but not RCK/p54, were downregulated immediately following activation of B cells with αIgM Abs, αIgM and αCD40 Abs and IL-4, or αCD40 Abs and IL-4. However, the mechanism of this  116  downregulation still needs to be investigated. I suspect that both secretion and degradation are involved. One could speculate that BCR signaling could induce ubiquitination and subsequent degradation of DCP1a and the P-body markers detected by the reference anti-GWB serum. Furthermore, it has previously been reported that both T and B lymphocytes release exosomes in response to activation signals (e.g. via Ag receptor), with particularly high levels of exosomes released by primary murine B lymphocytes stimulated αCD40 Abs and IL-4 189 (reviewed in Ref.  190  ). Exosomes have  been reported to contain GW182, but not DCP1a 191, and therefore GW182 could potentially be secreted via exosomes in response to BCR engagement, whereas DCP1a and the P-body markers detected by the reference anti-GWB serum could be secreted via a different type of vesicles (reviewed in Ref. 188). Due to time the constraint, I was not able to determine whether any of the P-body markers are either degraded or released via vesicles from primary murine B lymphocytes in response to BCR engagement. Moreover, primary murine B lymphocytes are the only type of B cells, in which I measured the total levels of P-body markers following stimulation via BCR. Thus, quantification of P-body markers after activation of other types of B cells would verify whether GWBs, GW182, and DCP1a, but not RCK/p54, are downregulated immediately following BCR engagement. 4.6.3. Analysis of P-bodies in activated B lymphocytes In this Chapter, I analyzed P-body expression in activated B cells and found that: Conclusion 5: Compared to naïve IgM+ B cells, B lymphocytes activated via their BCR contain elevated levels of P-body markers and a greater number of P-bodies.  117  Specifically, I showed that primary murine B lymphocytes activated for 24 hours with αIgM or with the combination of αIgM, αCD40, and IL-4, but not αCD40 and IL-4 alone, for 24 hours contained elevated levels of P-body markers and an increased number of P-bodies. As mentioned above, I was only able to measure the total levels of P-body markers following the stimulation of primary murine B lymphocytes via their BCRs, and not other types of B cells. Thus, thorough quantification of expression of P-body markers, as well as the number of P-bodies and total cumulative P-body area per cell, after activating multiple types of B cells could reinforce conclusion 5. Finally, because of the time constraint, I was not able to carry out the analysis of P-bodies in B lymphocytes following activation in vivo.  118  CHAPTER 5: THE ROLE OF P-BODIES IN MEMORY CD8+ T LYMPHOCYTES AND THEIR RECALL RESPONSE  5.1. Introduction Overall hypothesis: RANTES and IFN-γ effector mRNAs are stored in P-bodies of memory CD8+ T lymphocytes and are directed for translation at the immune synapse during the recall response (Illustration 1.1). •  Hypothesis 2: Memory CD8+ T lymphocytes contain more P-bodies than naïve T cells, and P-bodies in memory cells store RANTES and IFN-γ mRNAs. o Goal 2.1: Compare total expression levels, cumulative area, and numbers of P-bodies in naïve, activated, effector, and memory CD8+ T lymphocytes. o Goal 2.2: Identify the localization of RANTES and IFN-γ mRNAs relative to P-bodies in naïve, activated, effector, and memory CD8+ T lymphocytes.  •  Hypothesis 3: Upon activation of a lymphocyte, P-bodies are recruited to the immune synapse, where effector mRNAs are released for translation. o Goal 3.2: Test whether P-bodies are localized to the immune synapse in naïve, effector, and memory CD8+ T lymphocytes. In Chapter 4, I established that T and B lymphocytes contain P-bodies. Therefore,  it was possible to address hypothesis 2 and compare P-bodies at various stages of differentiation following activation of naïve CD8+ T lymphocytes. To this end, I have generated effector and memory CD8+ T lymphocytes in vitro according to the protocols developed in G.M. Griffith’s lab 178 and T.H. Watts’ lab 52,  119  respectively. In addition, as has been done in S.M. Russell’s lab 50, I utilized mature BMDCs instead of total splenocytes and imaged the interaction of Ag-specific T cells with mature BMDCs loaded with the peptide Ag in order to visualize the immune synapse and the first asymmetric division. In this model (Illustration 5.1), naïve CD8+ T lymphocytes are isolated from OT-I mice that are transgenic for TCR recognizing the ovalbumin (OVA) peptide OVA257-264 (SIINFEKL) on the MHC I molecule H-2Kb. Coculture with mature BMDCs that present SIINFEKL on H-2Kb activates these naïve CD8+ T lymphocytes. Addition of IL-2 to the co-culture simulates help from TH cells, allowing naïve cells to achieve full activation and rapidly proliferate. Activated CD8+ T lymphocytes are collected 48 hours later and separated into two flasks that are either (1) cultured with IL-2 for 3 extra days to induce differentiation into mature effector CD8+ T lymphocytes, or (2) cultured with IL-15 for 10 extra days to induce differentiation into mature memory CD8+ T lymphocytes. In this chapter, I demonstrate that compared to naïve CD8+ T lymphocytes from OT-I mice, activated T cells and effector T cells have elevated levels of P-body markers and a greater number of P-bodies. In contrast, memory T cells have similar numbers of Pbodies as naïve cells, but contain large GWBs and RCK/p54 granules. Furthermore, even though RANTES mRNA is highly expressed in effector and memory OT-I CD8+ T lymphocytes, it is diffusely distributed in the cytoplasm. On the other hand, IFN-γ mRNA is expressed in activated, effector, and memory cells and co-localizes with GWBs and RCK/p54 granules. Moreover, P-bodies partially dissociate and accumulate in proximity of the immune synapse in naïve, effector, and memory cells. Finally, GW182 seems to be distributed asymmetrically during the first division of naïve OT-I CD8+ T lymphocytes,  120  and from Jenkins, M.R. et al. Immunity (2009)  Illustration 5.1. Method outline for generating activated, effector, and memory CD8+ T cells from naïve OT-I CD8+ T lymphocytes in vitro. Immature BMDCs were prepared by culturing BM from B6 mice (H-2b haplotype) in complete RPMI containing 20 ng/mL GM-CSF for 7 days. These cells were induced to differentiate into mature BMDCs by addition of 100 ng/mL LPS and culturing overnight. On the next day, mature BMDCs were loaded with the OVA257-264 peptide SIINFEKL and mixed with naïve OT-I CD8+ T cells that recognize SIINFEKL on H2Kb. Following a 2-day incubation in presence of IL-2, these naïve cells became activated OT-I CD8+ T cells. To induce differentiation into effector OT-I CD8+ T cells, these activated cells were cultured for an additional 3 days in presence of IL-2. To differentiate into memory OT-I CD8+ T cells, the activated cells were cultured for an additional 10 days in presence of IL-15.  121  but only if the cell is attached to a DC.  5.2. Phenotypic and functional characterization of naïve, activated, effector, and memory OT-I CD8+ T lymphocytes 5.2.1. Naïve, activated, effector, and memory OT-I CD8+ T lymphocytes express correct surface markers Following isolation, naïve OT-I CD8+ T lymphocytes were highly enriched (>95%) CD8+ T cells (CD3+ CD8+) (Figure 5.1). The naïve phenotype of these cells was confirmed by their small size, by their low expression of CD25, the heavy (activated cellassociated) isoform of CD43, CD44, CD69, CD122, and KLRG1, and by their high expression of CD27, CD62L, and CD127. In addition, memory-phenotype cells (CD44High and CD122High) were successfully depleted during isolation. Activated OT-I CD8+ T lymphocytes were much larger than naïve cells, had reduced levels of cell surface CD3 (evidence of recent TCR engagement) and CD62L, and had increased expression of the activation markers CD25, heavy isoform of CD43, CD44, and CD69. This indicates that the naïve OT-I CD8+ T lymphocytes were successfully activated. Effector OT-I CD8+ T lymphocytes were large, had increased expression of CD3 compared to activated T cells, continued to express the activation markers CD25, the heavy isoform of CD43, CD44, and CD69, and had reduced expression of CD62L and CD127. Thus, these putative effector OT-I CD8+ T lymphocytes displayed the correct phenotype.  122  123  Figure 5.1. Naïve, activated, effector, and memory OT-I CD8+ T lymphocytes display correct phenotype. Size (FSC) and granularity (SSC), as well as surface expression of CD3, CD8, CD25, CD27, CD43, CD43 (activated cell-associated heavy isoform detected by mAb clone 1B11), CD44, CD62L, CD69, CD122,  124  CD127, and KLRG1 were analyzed on naïve, activated, effector, and memory OT-I CD8+ T cells by flow cytometry using the same settings and following staining with the same Ab master mixes. Results are representative of 3-5 independent experiments.  125  Memory OT-I CD8+ T lymphocytes were approximately the same size as naïve cells, but had increased granularity (SSC), as reported previously 52. Furthermore, these memory T cells expressed high levels of CD3, CD122, and CD127, intermediate levels of CD25 and CD44, and low levels of the activation marker CD69. Clearly distinguishable CD62LHigh (TCM) and CD62LLow (TEM) subsets of memory cells were present in this culture, as well as a small subset of cells expressing intermediate levels of the heavy isoform of CD43. These observations mirror the results seen in vivo 55 and confirm that this cell population was comprised mainly of memory OT-I CD8+ T lymphocytes. In addition, the activated, effector, and memory OT-I CD8+ T lymphocytes remained pure populations of CD8+ T cells (CD3+ CD8+). None of the cells in these populations expressed KLRG1, the marker of SLECs, as was previously reported for effector cells differentiated in present of IL-2 178. Overall, based on cell size and the expression of various surface markers, highly enriched populations of naïve, activated, effector, and memory OT-I CD8+ T lymphocytes were obtained. In previous published studies, only expression of the CD25, CD44, CD69, and CD62L markers was characterized for OT-I CD8+ T cells activated with SIINFEKL-loaded BMDCs 50. Therefore, documenting the expression of many of these surface markers is a novel result that further validates the protocol described in Illustration 5.1 for preparing these different cell populations.  126  5.2.2. Naïve, activated, effector, and memory OT-I CD8+ T lymphocytes exhibit correct functional properties To assess whether effector and memory OT-I CD8+ T lymphocytes retain their functional capabilities, I assayed their cytotoxic ability, proliferation in response to TCR engagement, and survival. As observed previously both in vitro and in vivo 28, 178, effector OT-I CD8+ T lymphocytes were highly cytotoxic when co-cultured with EL4 target cells that present SIINFEKL on H-2Kb (Illustration 5.2). These cytotoxic T cells killed the majority of EL4 cells within 4 hours when cultured at an effector to target (E : T) ratio of 10 (Figure 5.2A). On the other hand, memory cells were weakly cytotoxic and, as reported before 28, 52  , only killed ~9% of the target cells in 4 hours at E : T ratio of 10 (Figure 5.2B). Still, it  is important to keep in mind that the same memory CD8+ T cells that are weakly cytotoxic in vitro are able to induce the death of target cells in vivo 4 hours following adoptive transfer almost as efficiently as effector cells 28. I further evaluated the cytotoxic potential of effector and memory OT-I CD8+ T lymphocytes by culturing these cell populations with the target cells for prolonged periods of time (24 or 48 hours) at a lower E : T ratios of 0.3 (Illustration 5.3). The in vitro cytotoxicity of memory OT-I CD8+ T lymphocytes was more similar to that of effector cells at 24 and 48 hours than at 4 hours (Figure 5.2C and Figure 5.2D), suggesting that memory cells were able to quickly differentiate into secondary effector cells with high cytotoxic potential, a characteristic of memory CD8+ T cells undergoing the recall phase (Figure 1.1). In contrast to effector and memory OT-I CD8+ T lymphocytes, naïve OT-I CD8+ T cells did not display any  127  Illustration 5.2. Method outline for cytotoxicity assay with effector or memory OT-I CD8+ T lymphocytes and CellTracker Orange-labeled SIINFEKL-loaded EL4 target cells. EL4 target cells (H-2b haplotype) were labeled with CellTraker Orange, loaded with the OVA257-264 peptide SIINFEKL, and mixed with either effector or memory OT-I CD8+ T cells that recognize SIINFEKL on H2Kb at effector to target ratios (E : T) of 10, 3, 1, 0.3, 0.1 in triplicate. Naïve OT-I CD8+ T cells were assayed in parallel as a control. Following a 4-hour incubation, cells were stained with 7-AAD and analyzed by flow cytometry. Specific cytotoxicity was calculated by subtracting the percent of dead EL4 cells (CellTracker Orange+ 7-AAD+) without OT-I CD8+ T cells from the percent of dead EL4 cells with OT-I CD8+ T cells for each condition.  128  Illustration 5.3. Method outline for a combined assay to analyze long-term cytotoxicity, proliferation, and survival of effector or memory OT-I CD8+ T lymphocytes via co-culture with CellTracker Orange-labeled SIINFEKL-loaded EL4 target cells. EL4 target cells (H-2b haplotype) were labeled with CellTraker Orange, loaded with the OVA257-264 peptide SIINFEKL, and mixed with CFSE-labeled effector or memory OT-I CD8+ T cells that recognize SIINFEKL on H-2Kb at effector to target ratio (E : T) of 0.3. Naïve OT-I CD8+ T cells were assayed in parallel as a control. Following a 24-hour or a 48-hour incubation with or without IL-2, cells were stained with 7-AAD and analyzed by flow cytometry. Specific cytotoxicity was calculated by subtracting the percent of dead EL4 cells (CellTracker Orange+ 7-AAD+) without OT-I CD8+ T cells from the percent of dead EL4 cells with OT-I CD8+ T cells for each condition. Proliferation was analyzed in live OT-I CD8+ T cells (CFSE+ CellTracker Orange- 7-AAD-). The percent of surviving OT-I CD8+ T cells was calculated by subtracting the percent of dead OT-I CD8+ T cells (CFSE+ CellTracker Orange- 7-AAD+) from 100%.  129  Figure 5.2. In contrast to naïve cells, effector and memory OT-I CD8+ T lymphocytes display cytotoxicity towards EL4 target cells that present SIINFEKL on H-2Kb. Effector (A, C) or memory (B, D) OT-I CD8+ T cells were incubated with CellTraker Orange-labeled SIINFEKL-loaded EL4 target cells either at effector to target ratios (E : T) of 10, 3, 1, 0.3, 0.1 in triplicate for 4 hours (A-B), or at E : T = 0.3 with or without IL-2 for 24 or 48 hours (C-D). At the end of the incubation period, the cells were stained with 7-AAD, and analyzed by flow cytometry. Naïve OT-I CD8+ T cells were assayed in parallel as a control. Specific cytotoxicity was calculated by subtracting the percent of dead EL4 cells (CellTracker Orange+ 7-AAD+) without OT-I CD8+ T cells from the percent of dead EL4 cells with OT-I CD8+ T cells for each condition. (A-B) Mean ± SD. Results are representative of 2-3 independent experiments. (C-D) Mean ± SEM. Results are from 2 independent experiments.  130  cytotoxicity at 4, 24, or 48 hours, as expected for naïve cells activated by TCR engagement in the absence of co-stimulation (reviewed in Ref. 45). CFSE dilution analysis of T cell proliferation (Illustration 5.3) showed that both naïve and memory OT-I CD8+ T lymphocytes did not proliferate in response to IL-2 stimulation and/or TCR engagement for 24 hours (Figure 5.3A and Figure 5.3C). However, effector cells proliferated 1-2 times regardless of activation conditions (Figure 5.3B), suggesting that their proliferation was due to the IL-2 that had been present in the culture for the 5 previous days (Illustration 5.1). The differences between naïve and memory OT-I CD8+ T lymphocytes became apparent at 48 hours following activation: IL-2 induced vigorous proliferation of memory cells, but not naïve cells (Figure 5.4A and Figure 5.4C). Furthermore, TCR engagement for 48 hours regardless of IL-2 stimulation induced robust proliferation of both naïve and memory OT-I CD8+ T lymphocytes, with memory cells proliferating slightly faster. This observation is in agreement with the previous in vivo report showing robust proliferation of naïve CD8+ T cells in response to TCR engagement without co-stimulation that does not lead to cytotoxicity or effector cytokine production 81. Again, effector cells proliferated several times in 48 hours regardless of the activation conditions (Figure 5.4B), probably in response to IL-2 present in culture during their differentiation. However, both IL-2 stimulation and TCR engagement augmented the proliferation of effector OT-I CD8+ T lymphocytes in 48 hours. In summary, both naïve and memory OT-I CD8+ T lymphocytes displayed correct proliferative properties, whereas the proliferation of effector OT-I CD8+ T lymphocytes was difficult to assess due to IL-2 used for their differentiation.  131  Figure 5.3. In contrast to effector cells that were previously cultured with IL-2, naïve and memory OT-I CD8+ T lymphocytes do not proliferate in 24 hours following TCR engagement and/or stimulation with IL-2. 132  CFSE-labeled naïve (A), effector (B), or memory (C) OT-I CD8+ T cells were incubated with CellTraker Orange-labeled SIINFEKL-loaded EL4 target cells at effector to target ratio (E : T) of 0.3 with or without IL-2 for 24 hours. At the end of the incubation period, the cells were stained with 7-AAD, and analyzed by flow cytometry. Each type of OT-I CD8+ T cells was assayed alone or with EL4 cells that did not receive SIINFEKL in parallel as controls. Proliferation was analyzed in live OT-I CD8+ T cells (CFSE+ CellTracker Orange- 7-AAD-). Results are representative of 2-3 independent experiments.  133  Figure 5.4. In contrast to naïve cells, effector and memory OT-I CD8+ T lymphocytes can be induced to proliferate in 48 hours via stimulation with IL-2.  134  CFSE-labeled naïve (A), effector (B), or memory (C) OT-I CD8+ T cells were incubated with CellTraker Orange-labeled SIINFEKL-loaded EL4 target cells at effector to target ratio (E : T) of 0.3 with or without IL-2 for 48 hours. At the end of the incubation period, the cells were stained with 7-AAD, and analyzed by flow cytometry. Each type of OT-I CD8+ T cells was assayed alone or with EL4 cells that did not receive SIINFEKL in parallel as controls. Proliferation was analyzed in live OT-I CD8+ T cells (CFSE+ CellTracker Orange- 7-AAD-). Results are representative of 2-3 independent experiments.  135  Finally, in addition to being able to survive for 10 days in culture with the homeostatic turnover cytokine IL-15, as expected for memory cells (reviewed in Refs. 22, 33, 38, 53, 79, 80  ), the majority of memory OT-I CD8+ T lymphocytes were also able to  survive for 48 hours in absence of both IL-2 stimulation and TCR engagement (Figure 5.5C). In contrast, even though effector cells were not able to survive for 48 hours without IL-2 stimulation or TCR engagement, either one of these stimuli could protect effector cells from apoptosis (Figure 5.5B). On the other hand, naïve OT-I CD8+ T lymphocytes were only able to survive for 48 hours following TCR engagement, and IL-2 stimulation could not protect them from death (Figure 5.5A), as expected for naïve cells (reviewed in Refs. 22, 33, 38, 53, 79, 80). Overall, the survival ability of naïve, effector, and memory OT-I CD8+ T lymphocytes prepared according to the protocol in Illustration 5.1 was in agreement with the survival of naïve, effector, and memory CD8+ T lymphocytes in vivo. In conclusion, the above analysis confirms that mature populations of effector and memory OT-I CD8+ T lymphocytes were successfully prepared to address Hypotheses 2 and 3 of this thesis. Moreover, the above analysis of cytotoxic ability, proliferation in response to TCR engagement, and survival of effector and memory OT-I CD8+ T lymphocytes prepared according to Illustration 5.1, to my knowledge has not been done previously, including the study that used a similar method 50. Therefore, characterization of the functional properties of effector and memory OT-I CD8+ T lymphocytes in this study could aid future studies utilizing the method outlined in Illustration 5.1.  136  Figure 5.5. In contrast to naïve and effector cells, memory OT-I CD8+ T lymphocytes are able to survive up to 48 hours without TCR engagement and stimulation with IL-2. CFSE-labeled naïve (A), effector (B), or memory (C) OT-I CD8+ T cells were incubated with CellTraker Orange-labeled SIINFEKL-loaded EL4 target cells at effector to target ratio (E : T) of 0.3 with or without IL-2 for 48 hours. At the end of the incubation period, the cells were stained with 7-AAD, and analyzed by flow cytometry. Each type of OT-I CD8+ T cells was assayed alone or with EL4 cells that did not receive SIINFEKL in parallel as controls. The percent of surviving OT-I CD8+ T cells was calculated by subtracting the percent of dead OT-I CD8+ T cells (CFSE+ CellTracker Orange- 7-AAD+) from 100%. Mean ± SEM. Results are from 2 independent experiments.  137  5.3. Comparison of P-bodies in naïve, activated, effector, and memory OT-I CD8+ T lymphocytes When I compared the expression and localization of GWBs and the P-body markers GW182, RCK/p54, and DCP1a in naïve, activated, effector, and memory OT-I CD8+ T lymphocytes by flow cytometry and confocal microscopy, I found that compared to naïve cells, activated and effector cells had elevated total levels of P-body markers (Figure 5.6A and Figure 5.6C), a greater number of P-bodies (Figure 5.6B and Figure 5.6D), and an increased cumulative P-body area (Figure 5.6B and Figure 5.6E). In contrast, memory OT-I CD8+ T lymphocytes had similar numbers of P-bodies as naïve cells (Figure 5.6B and Figure 5.6D), but contained increased total levels of GWBs and RCK/p54 (Figure 5.6A and Figure 5.6C), as well as elevated cumulative area of GWBs and RCK/p54 granules (Figure 5.6B and Figure 5.6E). Upon detailed analysis, it became apparent that activated, effector, and memory OT-I CD8+ T lymphocytes contain large (> 0.7 µm) GWBs and RCK/p54 granules that are nearly absent in naïve cells. In addition, activated and effector OT-I CD8+ T lymphocytes contained large (> 0.6 µm) DCP1a granules, whereas these granules were essentially absent in both naïve and memory cells (Figure 5.6F). In Chapter 4, I found that both GWBs and RCK/p54 granules localize to the same subset of P-bodies in T lymphocytes. Consequently, memory OT-I CD8+ T lymphocytes contained large GWB+ RCK/p54+ granules. The finding that memory OT-I CD8+ T lymphocytes, but not naïve T cells, had large GWB+ RCK/p54+ granules, suggests that these P-bodies could contain memory cell-specific mRNAs that are “on stand-by” for the rapid recall response.  138  139  Figure 5.6. Memory OT-I CD8+ T lymphocytes contain large GWBs and RCK/p54 granules that are absent in naïve cells, but contain similar numbers of P-bodies. Naïve, activated, effector, and memory OT-I CD8+ T cells were fixed in 4% PFA and stained in suspension with 1º Abs specific for GWBs, GW182, RCK/p54, or DCP1a and α-tubulin, followed by 2º Abs conjugated to AlexaFluor488 (P-bodies, green) or AlexaFluor647 (α-tubulin, red). (A) ~75% of the cells were analyzed by flow cytometry to analyze expression levels of P-body markers and demonstrate consistency of staining on a population level. Note, FSC demonstrates relative cell size distribution. (B) ~25% of the cells were injected into mounting medium with DAPI (blue) and visualized by confocal microscopy. Results in (A) and (B) are representative of 4-5 independent experiments. (C) Total levels of P-body markers that are based on geometric MFI measured by flow cytometry from 4 (effector cells) or 5 (naïve, activated, and memory cells) independent experiments. Mean ± SEM. (D-F) Number of granules (D), cumulative granule area per cell (E), and granule size distribution (F) were quantified based on >120 cells from 3 independent experiments. Mean ± SD. *p < 0.05, **p < 0.001, ***p < 0.0001.  140  5.4. Analysis of mRNA localization to P-bodies in naïve, activated, effector, and memory OT-I CD8+ T lymphocytes 5.4.1. Poly(A) mRNA is located in the nucleus and diffusely in the cytoplasm of naïve, activated, effector, and memory OT-I CD8+ T lymphocytes Even though P-bodies in other cell types have been reported not to contain poly(A) mRNA (reviewed in Refs. 106, 192), I assessed the localization of poly(A) mRNA in naïve, activated, effector, and memory OT-I CD8+ T lymphocytes using FISH. Flow cytometry showed that each cell population contained poly(A) mRNA (Figure 5.7A), but that activated and effector OT-I CD8+ T lymphocytes contained more poly(A) mRNA than naïve and memory cells (Figure 5.7B), as expected from their larger size. As in CTLL2 cells (Figure 3.2), poly(A) mRNA was located in the nucleus and in the cytoplasm of all cell populations examined (Figure 5.7C). Specifically, I observed poly(A) mRNA in regions of the nucleus that were poorly stained with DAPI and which are likely to be euchromatin. I also found that poly(A) mRNA was distributed diffusely in the cytoplasm closer to the outer rim of the cell, but was not localized to discrete cytoplasmic granules. In addition, poly(A) mRNA was not co-localized with P-body markers (data not shown). This observation is consistent with the previous reports that Pbodies do not contain poly(A) mRNA. 5.4.2. RANTES mRNA is highly expressed and diffusely distributed in the cytoplasm of effector and memory OT-I CD8+ T lymphocytes In order to analyze the expression and localization of RANTES mRNA in naïve, activated, effector, and memory OT-I CD8+ T lymphocytes, I first determined the optimal probe concentration that would yield the greatest difference in staining between the  141  Figure 5.7. In naïve, activated, effector, and memory OT-I CD8+ T lymphocytes, poly(A) mRNA is located in the nucleus and distributed diffusely in the cytoplasm, but not in cytoplasmic granules. Fixed naïve, activated, effector, and memory OT-I CD8+ T cells were stained in suspension with Cy3oligo-d(T) probe that detects poly(A) mRNA (red). (A-B) ~75% of the cells were analyzed by flow  142  cytometry to analyze poly(A) mRNA expression on a population level in each cell type relative to a “no probe” control (A) or to naïve cells (B). Note, FSC demonstrates relative cell size distribution. (C) ~25% of the cells were injected into mounting medium with DAPI (blue) and visualized by confocal microscopy. Results are representative of 2 independent experiments.  143  RANTES mRNA probe and the Scrambled control probe. To do this, I used memory cells, which are known to highly express RANTES mRNA. 5, 6 I found that memory OT-I CD8+ T lymphocytes contain high amount of RANTES mRNA, and the highest FISH specificity was achieved with 1 pmol of both the RANTES and control probes (Figure 5.8A). Therefore, I have used 1 pmol of each probe per sample for all subsequent FISH experiments. Upon direct comparison, I found that naïve, activated, effector, and memory OT-I CD8+ T lymphocytes all expressed RANTES mRNA (Figure 5.8B), but that only effector and memory cells contained high amount of RANTES mRNA (Figure 5.8B, Figure 5.8C when normalized to cell size, and Figure 5.8D). Furthermore, the relative expression of RANTES mRNA among naïve, activated, effector, and memory OT-I CD8+ T lymphocytes was similar to that reported previously 6 (Figure 1.4B). RANTES mRNA did not form cytoplasmic granules, but instead was distributed diffusely in the cytoplasm (Figure 5.8D). Furthermore, RANTES mRNA was not colocalized with any of the P-body markers in activated, effector, and memory OT-I CD8+ T lymphocytes, but was sometimes associated with DCP1a granules in naïve cells (data not shown). Overall, the storage of RANTES mRNA in P-bodies of memory OT-I CD8+ T lymphocytes is not the mechanism of RANTES mRNA translational repression. Therefore, understanding how RANTES mRNA is translationally repressed in memory CD8+ T cells is still an outstanding question in immunology. 5.4.3. IFN-γ mRNA localizes to GWBs and RCK/p54 granules in activated, effector, and memory OT-I CD8+ T lymphocytes Flow cytometry analysis confirmed that in contrast to naïve cells, activated, effector, and memory OT-I CD8+ T lymphocytes express IFN-γ mRNA 5, 7 (Figure  144  145  Figure 5.8. RANTES mRNA is highly expressed and diffusely distributed in the cytoplasm of effector and memory OT-I CD8+ T lymphocytes. Fixed naïve, activated, effector, and memory OT-I CD8+ T cells were stained in suspension via FISH with double-DIG- labeled LNA probes specific for either RANTES mRNA or a Scramble control, followed by the anti-DIG 2ºAb conjugated to rhodamine (red). (A-C) ~75% of the cells were analyzed by flow cytometry to analyze RANTES mRNA expression on a population level in each cell type relative to the Scramble control (A-B) or to naïve cells (C). Note, F-actin staining (in separate samples) and FSC demonstrate relative cell size distribution. (D) ~25% of the cells were injected into mounting medium with DAPI (blue) and visualized by confocal microscopy. Results are representative of 3 independent experiments.  146  5.9A) and that activated, effector, and memory OT-I CD8+ T lymphocytes expressed more IFN-γ mRNA than naïve cells (Figure 5.9B). The difference in signal intensity between staining with the IFN-γ mRNA probe and the control probe was not as high as for RANTES mRNA, an observation that agrees with published results showing that IFNγ mRNA is expressed at lower levels than RANTES mRNA in memory CD8+ T cells 5 (Figure 1.4A). I found that IFN-γ mRNA was located in the cytoplasm of activated, effector, and memory OT-I CD8+ T lymphocytes and was concentrated in discrete puncta (Figure 5.9C). Importantly, IFN-γ mRNA co-localized with GWBs and RCK/p54 granules, but not with DCP1a or GW182 granules, in memory OT-I CD8+ T lymphocytes (Figure 5.10 and data not shown). Moreover, the IFN-γ mRNA was also co-localized with GWBs, RCK/p54 granules, and some of the DCP1a granules, but not with GW182 granules, in activated and effector OT-I CD8+ T lymphocytes (Figure 5.10 and data not shown). These observations suggest that the GWB+ RCK/p54+ P-bodies in memory OT-I CD8+ T lymphocytes, as well as the GWB+ RCK/p54+ P-bodies and some of the GWB+ RCK/p54+ DCP1a+ P-bodies in activated and effector OT-I CD8+ T lymphocytes, contain IFN-γ mRNA. In conclusion, IFN-γ mRNA, but not RANTES mRNA, is stored in the GWB+ RCK/p54+ subset of P-bodies in memory CD8+ T lymphocytes, which could allow these cells to rapidly secrete IFN-γ during the recall phase. Furthermore, resting effector CD8+ T lymphocytes do not synthesize IFN-γ, but are able to produce IFN-γ within 30 minutes of TCR engagement 54. Therefore, IFN-γ mRNA in GWB+ RCK/p54+ P-bodies of resting activated and effector CD8+ T cells could enable these cells to rapidly secrete  147  Figure 5.9. IFN-γ mRNA is expressed and localized to cytoplasmic granules in activated, effector, and memory OT-I CD8+ T lymphocytes. Fixed naïve, activated, effector, and memory OT-I CD8+ T cells were stained in suspension via FISH with double-DIG- labeled LNA probes specific for either IFN-γ mRNA or a Scramble control, followed by the  148  anti-DIG 2ºAb conjugated to rhodamine (red). (A-B) ~75% of the cells were analyzed by flow cytometry to analyze IFN-γ mRNA expression on a population level in each cell type relative to the Scramble control (A) or to naïve cells (B). Note, FSC demonstrates relative cell size distribution. (C) ~25% of the cells were injected into mounting medium with DAPI (blue) and visualized by confocal microscopy. Results are representative of 2 independent experiments.  149  Figure 5.10. IFN-γ mRNA granules co-localize with GWBs in activated, effector, and memory OT-I CD8+ T lymphocytes. Fixed naïve, activated, effector, and memory OT-I CD8+ T cells were stained in suspension with the reference αGWB human serum 18033, and then via FISH with the double-DIG-labeled LNA probe specific for IFN-γ mRNA, followed by 2ºAbs conjugated to either Alexa Fluor 488 (GWBs, green) or rhodamine (IFN-γ mRNA, red). The cells were subsequently injected into mounting medium with DAPI (blue) and visualized by confocal microscopy. Results are representative of 2 independent experiments.  150  IFN-γ upon encountering a target cell during infection, whereas IFN-γ mRNA in GWB+ RCK/p54+ DCP1a+ P-bodies might be undergoing degradation.  5.5. Analysis of P-body localization to the immune synapse in naïve, effector, and memory OT-I CD8+ T lymphocytes In preliminary experiments, I observed that some of the GW182+ P-bodies accumulate at the model immune synapse formed by Jurkat human CD4+ T cells, primary murine CD8+ T lymphocytes, or in vivo-generated murine CD8+ memory T cells that contact αCD3 mAb and αCD28 mAb-coated beads (data not shown). However, the rate of bead-cell conjugate formation was low, making the data difficult to interpret. Therefore, I assessed P-body localization in naïve OT-I CD8+ T lymphocytes following immune synapse formation with SIINFEKL-loaded mature BMDCs and in effector and memory OT-I CD8+ T lymphocytes forming immune synapse with SIINFEKL-loaded EL4 target cells. As I observed in B cells (Chapter 4), GWBs, RCK/p54 granules, and DCP1a granules partially dissociated and accumulated in proximity of the immune synapse in naïve OT-I CD8+ T lymphocytes interacting with SIINFEKL-loaded mature BMDCs for 20 minutes, 1 hour, and 4 hours, even though the best polarization was observed at 1 hour following co-culture initiation (Figure 5.11A and data not shown). To describe partial dissociation in other words, P-bodies became less defined as discrete foci (become fainter and in some cases fragmented), and I detected diffuse cytoplasmic P-body staining in the vicinity of remaining P-bodies. Similarly to B lymphocytes (Figure 4.12F), DCP1a granules disappeared to a greater extent than other P-bodies. Furthermore, GW182 granules seemed to accumulate directly at the immune synapse, rather than in its 151  Figure 5.11. GWBs localize in proximity of the immune synapse and GW182 is located at the immune synapse formed by naïve OT-I CD8+ T lymphocytes with SIINFEKL-loaded mature BMDCs over 1 hour. Naïve OT-I CD8+ T cells were co-cultured with SIINFEKL-loaded mature BMDCs for 1 hour, fixed in 4% PFA, and stained with (A) 1º Abs specific for GWBs, Lck, and α-tubulin, followed by 2º Abs conjugated to Alexa Fluor 488 (GWBs, green), Alexa Fluor 568 (Lck, red), and Alexa Fluor 647 (α-tubulin, violet), or (B) αCD3-FITC (green) and 1º Abs specific for GW182 and α-tubulin, followed by 2º Abs conjugated to Alexa Fluor 568 (α-tubulin, violet) and Alexa Fluor 647 (GW182, green). The cells were mounted in medium containing DAPI (blue) and visualized by confocal microscopy. Results are representative of 3-4 independent experiments.  152  Figure 5.12. GWBs localize in proximity of the immune synapse formed by effector and memory OT-I CD8+ T lymphocytes with SIINFEKL-loaded EL4 target cells over 1 hour. Effector (A) or memory (B) OT-I CD8+ T cells were co-cultured with SIINFEKL-loaded EL4 target cells for 1 hour, fixed in 4% PFA, and stained with 1º Abs specific for GWBs, Lck, and α-tubulin, followed by 2º Abs conjugated to Alexa Fluor 488 (GWBs, green), Alexa Fluor 568 (Lck, red), and Alexa Fluor 647 (αtubulin, violet). The cells were mounted in medium containing DAPI (blue) and visualized by confocal microscopy. Results are representative of 3 independent experiments.  153  proximity (Figure 5.11B and data not shown). Similar observations were obtained for effector and memory OT-I CD8+ T lymphocytes that had been allowed to bind to SIINFEKL-loaded EL4 target cells for 20 minutes, 1 hour, and 4 hours (Figure 5.12 and data not shown). In conclusion, TCR-induced activation of naïve, effector, and memory OT-I CD8+ T lymphocytes leads to partial dissociation and disappearance of GWBs, as well as GW182, RCK/p54, and DCP1a granules. In addition, the remaining GWBs, as well as RCK/p54 and DCP1a granules accumulate in proximity of the model immune synapse after 1 hour, whereas the remaining GW182 granules are localized directly at the immune synapse. This raises the possibility that P-bodies could deliver mRNAs to the immune synapse for rapid translation.  5.6. Analysis of P-body distribution during the first asymmetric division of naïve OT-I CD8+ T lymphocytes on SIINFEKL-loaded mature BMDCs P-bodies could play an important role in asymmetric distribution of mRNAs, such as IL-7Rα mRNA, during the first asymmetric division of naïve CD8+ T cells, potentially affecting the developmental decision between differentiation into either effector of memory CD8+ T cells. In order to test whether P-bodies are distributed asymmetrically during the first division, I assessed the kinetics of the first division of naïve OT-I CD8+ T lymphocytes on SIINFEKL-loaded mature BMDCs via CFSE dilution assay. Divided cells could be observed at 34 hours, but not at 28 hours, following initiation of the coculture (Figure 5.13). Therefore, the first division of naïve OT-I CD8+ T lymphocytes takes place between 28 and 34 hours.  154  Figure 5.13. Naïve OT-I CD8+ T lymphocytes start dividing between 28 and 34 hours following activation by SIINFEKL-loaded mature BMDCs. 4 x 105 CFSE-labeled naïve OT-I CD8+ T cells were incubated with 4 x 105 SIINFEKL-loaded mature BMDCs (E : T = 1) in presence of IL-2 for 22, 28, 34, or 48 hours. At the end of the incubation period, the non-adherent cells were thoroughly washed off, stained with 7-AAD, and analyzed by flow cytometry. Proliferation was analyzed in live OT-I CD8+ T cells (CFSE+ 7-AAD-). Results are representative of 3-4 independent experiments.  155  I observed symmetric distribution of the surface markers CD3, CD8, CD25, CD43 (heavy isoform), CD44, CD62L, and CD69 in naïve OT-I CD8+ T cells that divided once, based on these cells belonging to the second CFSE peak (data not shown). This observation confirms findings published in the second report on asymmetric division of T cells 50 and contradicts the initial publication 47. Since there are only two reports published on this subject to date with opposing findings, it is important to note that my observations confirmed the results from S.M. Russell’s lab 50. To identify APC-associated T cells undergoing their first division, I searched for mitotic cells among naïve cell-BMDC conjugates that formed at 28 hours of co-culture. In such mitotic cells, I observed asymmetric distribution of the Par6 and Scribble polarity proteins, with Par6 accumulating in the distal daughter cell (Figure 5.14A and Figure 5.14C) and Scribble distributing towards the proximal daughter cell (Figure 5.14B), as reported previously 50. I found that P-bodies containing GWBs, RCK/p54, and DCP1a were polarized towards the BMDC at 28 hours of co-culture, even though the cells became larger and had more P-bodies (Figure 5.14B and data not shown). In contrast, GW182 accumulated at the APC-distal side of the OT-I CD8+ T cell at this time (Figure 5.14A). Moreover, GW182 seemed to retain polarization away from BMDC during the first division, and was thus enriched in the distal daughter cell (Figure 5.14A). In contrast, P-bodies containing GWBs, RCK/p54, and DCP1a did not seem to be distributed asymmetrically during cell division (Figure 5.14C and data not shown). If Pbodies are involved in asymmetric distribution of mRNAs, such as IL-7Rα mRNA, during the initial asymmetric division of T-cells, GW182 could mediate this function.  156  157  Figure 5.14. Localization of GWBs and GW182 right before and during the first asymmetric division of naïve OT-I CD8+ T lymphocytes on SIINFEKL-loaded mature BMDCs. Naïve OT-I CD8+ T cells were co-cultured with SIINFEKL-loaded mature BMDCs for 28 hours, fixed in 4% PFA, and stained with (A) 1º Abs specific for GW182 and Par6, followed by 2º Abs conjugated to Alexa Fluor 488 (GW182, green) and Alexa Fluor 568 (Par6, red), (B) 1º Abs specific for GWBs, Scribble, and α-tubulin, followed by 2º Abs conjugated to Alexa Fluor 488 (GWBs, green), Alexa Fluor 568 (Scribble, red), and Alexa Fluor 647 (α-tubulin, violet), or (C) 1º Abs specific for GWBs, Par6, and αtubulin, followed by 2º Abs conjugated to Alexa Fluor 488 (GWBs, green), Alexa Fluor 568 (Par6, red), and Alexa Fluor 647 (α-tubulin, cyan). The cells were mounted in medium containing DAPI (blue) and visualized by confocal microscopy. Results are representative of ≥ 40 non-mitotic cells and ≥ 5 mitotic cells from 3-4 independent experiments.  158  5.7. Discussion 5.7.1. Characterization of phenotype and functional properties of naïve, activated, effector, and memory OT-I CD8+ T lymphocytes In this Chapter, I established that: Conclusion 1: I adapted published protocols to generate naïve, activated, effector, and memory OT-I CD8+ T lymphocytes that have correct phenotype and functional properties. Exactly, I characterized the size, surface markers, and functional properties of naïve, activated, effector, and memory OT-I CD8+ T lymphocytes prepared as described in Illustration 5.1. The in vitro differentiation of effector and memory CD8+ T lymphocytes was carried out in vitro according to the protocols developed in G.M. Griffith’s lab 178 and T.H. Watts’ lab 52, respectively. However, I have modified the above protocols by using SIINFEKL-loaded mature BMDCs instead of total splenocytes for T-cell activation in order to visualize the immune synapse, the first asymmetric division, and to improve the purity of isolated T cells. Furthermore, during the course of this study, another report using a similar model was published by the S.M. Russell’s lab. 50  In their system, the authors also used SIINFEKL-loaded mature BMDCs to activate  naïve OT-I CD8+ T lymphocytes and differentiate them into effector and memory cells in vitro. The only difference between our systems is the method of BMDC maturation: in my model, BMDCs were matured with LPS (as suggested by P. Johnson’s lab and K. Harder’s lab at the University of British Columbia), whereas the authors of Ref. 50 utilized IL-4 to induce BMDC maturation.  159  Based on cell size (FSC), surface expression of CD3, CD8, CD25, CD27, CD43 (heavy isoform and total), CD44, CD62L, CD69, CD122, CD127, and KLRG1, cytotoxicity towards SIINFEKL-loaded EL4 target cells, proliferation, and survival, I concluded that the naïve, activated, effector, and memory OT-I CD8+ T lymphocytes that I generated had the correct phenotype and functional properties. The only exception is the lack of KLRG1 expression by effector cells, although KLRG1 is a marker of SLECs, which are the majority of effector cells in vivo (reviewed in Ref. 33, 38). However, it has been previously established that IL-2 protects effector CD8+ T cells from apoptosis and prevents KLRG1 expression on their surface. Therefore, the lack of KLRG1 expression on the surface of effector OT-I CD8+ T lymphocytes that I have generated is an expected phenotype. Nevertheless, considering their expression of other surface markers, high cytotoxic potential, and rapid death upon IL-2 withdrawal, effector CD8+ T lymphocytes were correctly differentiated from naïve OT-I CD8+ T cells. Only the expression of CD25, CD44, CD69, and CD62L markers was characterized on effector and memory OT-I CD8+ T cells differentiated via activation with SIINFEKL-loaded mature BMDCs previously 50, and the analysis of cytotoxic potential, proliferation, and survival in this model, to my knowledge, has not been done before. Therefore, documenting the expression of many of the above surface markers and characterizing the functional properties in this model could aid future studies utilizing the method outlined in Illustration 5.1. 5.7.2. Comparison of P-bodies in naïve, activated, effector, and memory OT-I CD8+ T lymphocytes In this Chapter, I also assessed hypothesis 2 and established that:  160  Conclusion 2: Compared to naïve CD8+ T cells, activated and effector CD8+ T lymphocytes contain elevated levels of P-body markers and a greater number of P-bodies. Conclusion 3: Memory CD8+ T lymphocytes have a similar number of P-bodies as naïve CD8+ T cells, but express higher levels of GWBs and RCK/p54 and contain large GWBs and RCK/p54 granules. Specifically, based on the MFI of P-body markers as determined by flow cytometry and the cumulative area of P-bodies per cell quantified from confocal microscopy images, I concluded that activated and effector CD8+ T lymphocytes express more GWBs, GW182, RCK/p54, and DCP1a than naïve cells, whereas memory CD8+ T lymphocytes only express higher total levels of GWBs and RCK/p54 than naïve CD8+ T cells. Furthermore, based on the number of P-bodies per cell quantified via confocal microscopy, activated and effector CD8+ T lymphocytes appear to contain more GWBs, as well as GW182, RCK/p54, and DCP1a granules than naïve cells, whereas memory CD8+ T lymphocytes contain similar numbers of all P-bodies as naïve CD8+ T cells. In addition, size distribution analysis showed that activated and effector CD8+ T lymphocytes contain large (> 0.6-0.7 µm) GWBs, as well as RCK/p54 and DCP1a granules, which are thought to be associated with mRNA turnover. Moreover, I found that memory CD8+ T lymphocytes contain large (> 0.7 µm) GWBs and RCK/p54 granules. I speculate that these large GWB+ RCK/p54+ P-bodies contain memory cellspecific mRNAs that are stored in such granules in order to facilitate rapid recall responses. However, as I discussed in Chapter 4, the light-based resolution limits of confocal microscopy (x-y and z resolution is ~200 nm and ~500 nm, respectively), prevent me from distinguishing one large P-body from several smaller ones that are 161  closer than the above resolution limits. Therefore, I could have underestimated the number of P-bodies and could have mistaken a group of small P-bodies for one large Pbody. The analysis of P-bodies by electron microscopy could be performed to address the above uncertainties. 5.7.3. Analysis of mRNA localization to P-bodies in naïve, activated, effector, and memory OT-I CD8+ T lymphocytes In this Chapter, I further assessed hypothesis 2 via co-localization analysis of poly(A), RANTES, and IFN-γ mRNAs with P-bodies and determined that: Conclusion 4: Poly(A) mRNA is located in what may be euchromatin regions of the nucleus and distributed diffusely in the cytoplasm, but does not co-localize with P-bodies in naïve, activated, effector, and memory CD8+ T lymphocytes. Conclusion 5: RANTES mRNA is highly expressed and diffusely distributed in the cytoplasm of effector and memory CD8+ T lymphocytes. Conclusion 6: IFN-γ mRNA is highly expressed and can be found in GWB+ RCK/p54+ P-bodies of activated, effector, and memory CD8+ T lymphocytes. The observation that P-bodies do not contain poly(A) mRNA is in agreement with data from other cell types (reviewed in Refs. 106, 192). However, the analysis of poly(A) mRNA localization in naïve, activated, effector, and memory CD8+ T lymphocytes, to my knowledge, has not been done previously. My observation that RANTES mRNA is distributed diffusely in the cytoplasm of effector and memory CD8+ T lymphocytes could be explained by its lack of P-body  162  targeting elements, such as miRNA binding sites or AREs. However, the mechanism of RANTES mRNA translational repression in these cells still needs to be delineated. Finally, because I found that translationally-repressed IFN-γ mRNA is located in GWB+ RCK/p54+ P-bodies of activated, effector, and memory CD8+ T lymphocytes, I propose the following model: miRNAs or stabilizing ARE-BPs (e.g. HuR) direct IFN-γ mRNA into GWB+ RCK/p54+ P-bodies for storage, whereas RANTES mRNA that lacks these P-body targeting elements is stored diffusely in the cytoplasm. The observation that IFN-γ mRNA is sometimes also found in GWB+ RCK/p54+ DCP1a+ P-bodies of activated and effector CD8+ T lymphocytes may imply that IFN-γ mRNA is directed into both in GWB+ RCK/p54+ and GWB+ RCK/p54+ DCP1a+ P-bodies in primary effector populations (SLECs and MPECs), but the proportion of IFN-γ mRNA in GWB+ RCK/p54+ DCP1a+ P-bodies is eventually degraded, and thus only IFN-γ mRNA in GWB+ RCK/p54+ P-bodies remains. Addressing the above speculations is one of the main future directions of this thesis. The conclusions that GWB+ RCK/p54+ P-bodies of activated, effector, and memory CD8+ T lymphocytes contain IFN-γ mRNA, whereas none of the P-bodies analyzed contain poly(A) mRNA or RANTES mRNA, could be strengthened via crosslinking and immunoprecipitation (CLIP) of P-body markers followed by the analysis of these mRNAs in cytoplasmic and P-body-associated fractions via quantitative PCR (qPCR). Moreover, the above conclusions need to be tested by the analysis of T cells prepared in vivo.  163  5.7.4. Evaluation of P-body localization to the immune synapse in T lymphocytes In Chapter, I assessed hypothesis 3 in T lymphocytes and found that: Conclusion 7: In naïve, effector, and memory CD8+ T lymphocytes, GWB+ RCK/p54+ and GWB+ RCK/p54+ DCP1a+ P-bodies partially dissociate and localize in proximity of the immune synapse, whereas GW182+ P-bodies localize at the immune synapse. Specifically, I found that, like in B cells, GWBs, RCK/p54, and DCP1a become less defined as discrete foci and localize in proximity of the immune synapse formed by naïve CD8+ T lymphocytes with SIINFEKL-loaded mature DCs and by effector and memory CD8+ T lymphocytes with SIINFEKL-loaded EL4 target cells, whereas GW182 moves directly to the immune synapse. These observations are consistent with the overall hypothesis that effector mRNAs are stored in P-bodies of memory CD8+ T lymphocytes and are directed for translation at the immune synapse during the recall response. The above conclusions are based on the confocal microscopy analysis of fixed cells, and real-time imaging of live cells could strengthen these conclusions. For example, expressing P-body markers tagged with a fluorescent protein in various T and B cells (e.g. via Amaxa electroporation), and then activating these cells via their Ag receptor, could provide initial data. It is a promising future direction to answer the question whether IFN-γ mRNA stored in GWB+ RCK/p54+ P-bodies of memory CD8+ T lymphocytes is transported towards the immune synapse by P-bodies and then released from P-bodies into the translationally-active pool. Moreover, the findings that IFN-γ mRNA is located in both GWB+ RCK/p54+ and GWB+ RCK/p54+ DCP1a+ P-bodies of effector CD8+ T 164  lymphocytes, and that both of these P-body subsets are localized in proximity of the immune synapse in effector cells, suggest that perhaps P-bodies could deliver effector mRNA towards the immune synapse in effector CD8+ T cells. Finally, since P-bodies move towards the model immune synapse in naïve CD8+ T lymphocytes as well, mobilization of mRNAs from P-bodies could also be an important step for full activation and/or fate specification of CD8+ T cells. Addressing these speculations is a key extension of the work presented in this thesis. 5.7.5. Assessment of P-body distribution during the first asymmetric division of naïve CD8+ T lymphocytes Finally, in this Chapter, via confocal microscopy I found that: Conclusion 8: GWB+ RCK/p54+ and GWB+ RCK/p54+ DCP1a+ P-bodies polarize towards the DC just before the first asymmetric division of naïve CD8+ T lymphocytes, whereas GW182+ P-bodies polarize away from the DC. Conclusion 9: GWB+ RCK/p54+ and GWB+ RCK/p54+ DCP1a+ P-bodies are weakly polarized during the first asymmetric division of naïve CD8+ T lymphocytes on the DC, whereas GW182+ P-bodies localize to the distal daughter cell. The asymmetric distribution of P-bodies prior to and/or the during the initial asymmetric division of T-cells, could be important for the correct localization of mRNAs involved in fate specification, such as IL-7Rα mRNA. Since both IL-7Rα mRNA 47 and GW182 localize to the distal daughter cell during the first asymmetric division of T-cells, GW182 could mediate the transport of IL-7Rα mRNA at the time of this division.  165  However, conclusion 9 is still preliminary, because I was not able to image sufficient number of cells to make this conclusion with high confidence. Nevertheless, the experimental set up for this part is robust, and thorough microscopy with image quantification could be used to support conclusion 9.  166  CHAPTER 6: DISCUSSION  6.1. Introduction In Chapter 1 I stated: Overall hypothesis: RANTES and IFN-γ effector mRNAs are stored in P-bodies of memory CD8+ T lymphocytes and are directed for translation at the immune synapse during the recall response (Illustration 1.1). •  Hypothesis 1: T and B lymphocytes contain P-bodies. o Goal 1.1: Establish whether human and murine T and B-cell lines, as well as primary murine CD4+ T, CD8+ T, and B lymphocytes contain GWBs and express the P-body markers GW182, RCK/p54, and DCP1a that are localized to discrete cytoplasmic foci. o Goal 1.2: Determine whether GWBs, GW182, RCK/p54, and DCP1a are localized to the same cytoplasmic granules.  •  Hypothesis 2: Memory CD8+ T lymphocytes contain more P-bodies than naïve T cells, and P-bodies in memory cells store RANTES and IFN-γ mRNAs. o Goal 2.1: Compare total expression levels, cumulative area, and numbers of P-bodies in naïve, activated, effector, and memory CD8+ T lymphocytes. o Goal 2.2: Determine the localization of RANTES and IFN-γ mRNAs relative to P-bodies in naïve, activated, effector, and memory CD8+ T lymphocytes.  •  Hypothesis 3: Upon activation of a lymphocyte, P-bodies are recruited to the immune synapse, where effector mRNAs are released for translation.  167  o Goal 3.1: Determine whether P-bodies are recruited to clustered IgM in primary murine B lymphocytes. o Goal 3.2: Test whether P-bodies are localized to the immune synapse in naïve, effector, and memory CD8+ T lymphocytes. In this thesis, I characterized P-bodies in T and B lymphocytes and addressed the potential role of P-bodies in immune memory by achieving the above goals. Below is the list of conclusions made throughout this thesis.  6.2. Main conclusions 6.2.1. Development of the protocol for dual analysis of proteins and/or mRNAs in lymphocytes by flow cytometry and confocal microscopy I developed a technique that proved to be a reliable method to stain lymphocytes in suspension and analyze the same sample by confocal microscopy to determine intracellular protein localization, as well as by flow cytometry to assess overall protein and/or mRNA expression levels (Chapter 3, Illustration 6.1). I have tested this method in various T and B-cell lines, and well as in primary T and B lymphocytes. Furthermore, the protocol works equally well in both low-throughput (individual tubes) and highthroughput (96-well plate) formats. Since a single staining procedure enables detection via two different methods, this protocol reduces variability, saves time, and economizes on reagents. The first detection method, flow cytometry, provides information on staining consistency, protein and/or mRNA expression levels, and rare events that could be missed if confocal microscopy were the only detection method. The second detection method, confocal microscopy, utilizes optical slicing and sequential imaging of  168  fluorophores, which can be used in conjunction with 3D-reconstruction, to provide high resolution of intracellular features. Finally, dual analysis is optional, and this staining  Illustration 6.1. Method outline for dual analysis of proteins and/or mRNAs in lymphocytes by flow cytometry and confocal microscopy. Lymphocytes (e.g. T cells) must be fixed in 4% PFA, permeabilized with SWB, incubated with blocking buffer containing MAXblock, BSA, and serum of the 2º Ab species, stained in suspension with 1º Abs (e.g. specific for GWBs), and then with 2º Abs conjugated to a fluorophore (e.g. Alexa Fluor 488, GWBs, green). Rhodamine-phalloidin (red) can be included into 2º Ab master mix to mark F-actin. ~25% of the cells should be injected into mounting medium with or without DAPI (blue) to visualize fine intracellular localization by confocal microscopy, and the remaining ~75% of the cells can be analyzed by flow cytometry to determine staining consistency and expression of proteins and/or mRNAs.  169  protocol can be used for either flow cytometry or confocal microscopy with very low background and excellent quality. Image cytometry is a novel technique that has recently been developed by Amnis (http://www.amnis.com), such that its products, FlowSight and ImageStreamX, combine the high speed of flow cytometers with the ability to capture images of individual cells. The technique described in this thesis combines the capabilities of modern flow cytometers, which enables the analysis of up to 20,000 cells per second, with the abilities of modern confocal microscopes, allowing to achieve the x-y and z resolution of as low as 200 nm and 500 nm, respectively, as well as to utilize sequential scanning that precludes fluorophores from affecting each other. Image cytometers cannot yet achieve such characteristics. However, the instruments like FlowSight and ImageStreamX provide images of the exact same cells that are analyzed, making it possible to relate the flow cytometry values to the images, whereas the technique presented here does not allow such comparison. Overall, I believe that the protocol for dual analysis of lymphocytes by flow cytometry and confocal microscopy presented here could be a useful tool for immunologists studying various aspects of lymphocyte biology. 6.2.2. Analysis of P-bodies in T and B lymphocytes In Chapter 4, I addressed hypothesis 1 and established that: Conclusion 1: T and B lymphocytes contain P-bodies. I found that human and murine T and B-cell lines, as well as primary murine  170  CD4+ T, CD8+ T, and B lymphocytes contain GWBs and express the P-body markers GW182, RCK/p54, and DCP1a, and that these P-body markers are concentrated in discrete cytoplasmic granules. Furthermore, I determined that: Conclusion 2: There are three main P-body subsets in T lymphocytes: GWB+ RCK/p54+, GWB+ RCK/p54+ DCP1a+, and GW182+ P-bodies. Conclusion 3: There are two main P-body subsets in B lymphocytes: GWB+ RCK/p54+ DCP1a+ and GW182+ P-bodies. Specifically, I found that P-bodies are heterogeneous in size and protein composition, but that the number of P-bodies and the cumulative area per cell for each Pbody marker is fairly constant in primary T and B lymphocytes. Furthermore, based on co-localization analysis (summarized in Illustration 6.2 for primary murine T and B cells), I determined that there are three main P-body subsets in T lymphocytes: GWB+ RCK/p54+ DCP1a+, GWB+ RCK/p54+, and GW182+ P-bodies, whereas that there are only two main P-body subsets in B lymphocytes: GWB+ RCK/p54+ DCP1a+ and GW182+ P-bodies. 6.2.3. Characterization of phenotype and functional properties of naïve, activated, effector, and memory OT-I CD8+ T lymphocytes In Chapter 5, I established that: Conclusion 4: I adapted published protocols to generate naïve, activated, effector, and memory OT-I CD8+ T lymphocytes that have correct phenotype and functional  171  Illustration 6.2. P-body subsets in primary T and B lymphocytes. Average number and protein composition of P-bodies in a representative (A) primary CD4+ T lymphocyte, (B) primary CD8+ T lymphocyte, and (C) primary B lymphocyte from WT mice.  172  properties. Based on cell size, surface markers, and functional properties of naïve, activated, effector, and memory OT-I CD8+ T lymphocytes prepared in vitro as described in Illustration 5.1, I concluded that effector and memory CD8+ T lymphocytes were correctly differentiated from naïve OT-I CD8+ T cells. Moreover, only the expression of CD25, CD44, CD69, and CD62L surface markers was characterized on effector and memory OT-I CD8+ T cells differentiated via activation with SIINFEKL-loaded mature BMDCs previously 50. Thus, documenting the expression of CD27, CD43 (heavy isoform and total), CD122, CD127, and KLRG1 surface markers and characterizing the cytotoxic potential, proliferation, and survival in this model could aid future studies utilizing the method outlined in Illustration 5.1. 6.2.4. Comparison of P-bodies in naïve, activated, effector, and memory OT-I CD8+ T lymphocytes In Chapter 5, I assessed hypothesis 2 and established that: Conclusion 5: Compared to naïve CD8+ T cells, activated and effector CD8+ T lymphocytes contain elevated levels of P-body markers and a greater number of P-bodies. Conclusion 6: Memory CD8+ T lymphocytes have a similar number of P-bodies as naïve CD8+ T cells, but express higher levels of GWBs and RCK/p54 and contain large GWBs and RCK/p54 granules. Specifically, I concluded that activated and effector CD8+ T lymphocytes express elevated levels of all P-body markers than naïve cells, whereas memory CD8+ T  173  lymphocytes only express higher elevated levels of GWBs and RCK/p54 than naïve cells. Furthermore, I found that activated and effector CD8+ T lymphocytes contain more GWBs, as well as GW182, RCK/p54, and DCP1a granules than naïve cells, whereas memory CD8+ T lymphocytes contain similar numbers of all P-bodies to naïve cells. In addition, I observed that activated, effector, and memory CD8+ T lymphocytes contain large GWB+ RCK/p54+ P-bodies, whereas activated and effector cells also contain large GWB+ RCK/p54+ DCP1a+ P-bodies. 6.2.5. Analysis of P-bodies in activated B lymphocytes In Chapter 4, I analyzed P-body expression in activated B cells and found that: Conclusion 7: Compared to naïve IgM+ B cells, B lymphocytes activated via their BCR contain elevated levels of P-body markers and a greater number of P-bodies. Specifically, I showed that primary murine B lymphocytes activated for 24 hours with αIgM or with the combination of αIgM, αCD40, and IL-4, but not αCD40 and IL-4 alone, for 24 hours upregulate levels of P-body markers and contain an increased number of P-bodies. Thus: Conclusion 8: Ag receptor-induced activation of T and B lymphocytes leads to an increase in the total levels of P-body markers and an increase in the number of P-bodies. 6.2.6. Analysis of mRNA localization to P-bodies in naïve, activated, effector, and memory OT-I CD8+ T lymphocytes In Chapter 5, I assessed hypothesis 2 via co-localization analysis of poly(A), RANTES, and IFN-γ mRNAs with P-bodies and determined that:  174  Conclusion 9: Poly(A) mRNA is located in what may be euchromatin regions of the nucleus and distributed diffusely in the cytoplasm, but does not co-localize with P-bodies in naïve, activated, effector, and memory CD8+ T lymphocytes. Conclusion 10: RANTES mRNA is highly expressed and diffusely distributed in the cytoplasm of effector and memory CD8+ T lymphocytes. Conclusion 11: IFN-γ mRNA is highly expressed and can be found in GWB+ RCK/p54+ P-bodies of activated, effector, and memory CD8+ T lymphocytes. Consistent with data from other cell types (reviewed in Refs. 106, 192), I found that P-bodies do not contain poly(A) mRNA. Moreover, because I detected IFN-γ mRNA, but not RANTES mRNA, in GWB+ RCK/p54+ P-bodies of memory CD8+ T lymphocytes, I propose the following model: miRNAs or stabilizing ARE-BPs direct IFN-γ mRNA into GWB+ RCK/p54+ P-bodies for storage, whereas RANTES mRNA that lacks the P-body targeting elements, such as miRNA binding sites or AREs, is stored diffusely in the cytoplasm (Illustration 6.3). In addition, I concluded that IFN-γ mRNA is localized in GWB+ RCK/p54+ and some of the GWB+ RCK/p54+ DCP1a+ P-bodies in activated and effector CD8+ T lymphocytes. Furthermore, I speculated that the storage of IFN-γ mRNA in GWB+ RCK/p54+ P-bodies of resting activated and effector CD8+ T cells could enable these cells to rapidly produce IFN-γ within 30 minutes of encountering an infected cell, whereas IFN-γ mRNA in GWB+ RCK/p54+ DCP1a+ P-bodies is undergoing degradation. 6.2.7. Evaluation of P-body localization to the immune synapse in T and B lymphocytes In Chapter 4, I tested hypothesis 3 in B lymphocytes and found that:  175  Illustration 6.3. Proposed model for the storage of effector cytokine mRNAs in memory CD8+ T lymphocytes. RANTES mRNA is stored diffusely in the cytoplasm of memory CD8+ T cells via a yet-to-be-defined mechanism, while IFN-γ mRNA is stored in GWB+ RCK/p54+ P-bodies, allowing memory cells to rapidly secrete RANTES and IFN-γ effector cytokines during the recall phase.  176  Conclusion 12: In B lymphocytes, P-bodies partially dissociate in response to BCR cross-linking, and the remaining GWB+ RCK/p54+ DCP1a+ P-bodies localize in proximity of the model immune synapse, whereas remaining GW182+ P-bodies localize at the model immune synapse. In Chapter 5, I assessed hypothesis 3 in T lymphocytes and found that: Conclusion 13: In naïve, effector, and memory CD8+ T lymphocytes, GWB+ RCK/p54+ and GWB+ RCK/p54+ DCP1a+ P-bodies partially dissociate and localize in proximity of the immune synapse, whereas GW182+ P-bodies localize at the immune synapse. Thus: Conclusion 14: In T and B lymphocytes, GWB+ RCK/p54+ and/or GWB+ RCK/p54+ DCP1a+ P-bodies partially dissociate and localize in proximity of the immune synapse, whereas GW182+ P-bodies localize at the immune synapse. Consequently, I speculated that in T and B lymphocytes, specific mRNAs are transported towards the immune synapse by P-bodies and then released from P-bodies into the translationally-active pool. 6.2.8. Assessment of P-body distribution during the first asymmetric division of naïve CD8+ T lymphocytes Finally, in Chapter 5, via confocal microscopy I found that: Conclusion 15: GWB+ RCK/p54+ and GWB+ RCK/p54+ DCP1a+ P-bodies polarize towards the DC just before the first asymmetric division of naïve CD8+ T lymphocytes, whereas GW182+ P-bodies polarize away from the DC.  177  Conclusion 15: GWB+ RCK/p54+ and GWB+ RCK/p54+ DCP1a+ P-bodies are weakly polarized during the first asymmetric division of naïve CD8+ T lymphocytes on the DC, whereas GW182+ P-bodies localize to the distal daughter cell. Subsequently, I speculated that the asymmetric distribution of P-bodies prior to and/or during the initial asymmetric division of CD8+ T lymphocytes could be an important mechanism for the correct localization of mRNAs involved in fate specification, such as IL-7Rα mRNA. I further proposed that GW182 could mediate the transport of IL-7Rα mRNA at the time of this first division.  6.3. Future directions Even though the time constraint prevented me from characterizing the specific functions of GWB+ RCK/p54+ DCP1a+, GWB+ RCK/p54+, and GW182+ P-bodies in T lymphocytes and determining the significance of DCP1a being present in most of the GWB+ RCK/p54+ P-bodies in B lymphocytes, I believe understanding these phenomena is an important future direction. As I discussed in Chapter 4, one could speculate that GW182+ P-bodies are aggregates of miRISCs that are subsequently sorted for storage into GWB+ RCK/p54+ P-bodies, degradation in GWB+ RCK/p54+ DCP1a+ P-bodies, or secretion (e.g. via exospores). Moreover, both GWB+ RCK/p54+ P-bodies and GWB+ RCK/p54+ DCP1a+ P-bodies potentially contain mRNAs delivered in an ARE-dependent and miRNA-independent manner. There is a possibility that P-bodies with different protein compositions could have distinct functional properties, such as translational repression or mRNA degradation. Furthermore, as I speculated in Chapter 4, both GWB+ RCK/p54+ and GW182+ P-bodies could play a role in translational repression, even  178  though via different mechanisms, whereas GWB+ RCK/p54+ DCP1a+ P-bodies are potentially involved in mRNA degradation. Characterizing the specific roles of GWB+ RCK/p54+ DCP1a+, GWB+ RCK/p54+, and GW182+ P-bodies could be achieved via CLIP of P-body markers in T and B lymphocytes followed by the analysis of specific mRNAs in cytoplasmic and P-bodyassociated fractions via qPCR. Moreover, microfluidics technologies, such as Fluidigm (http://www.fluidigm.com), can provide qPCR data on hundreds of genes from a single sample. If resources allow, CLIP of P-body markers followed by mRNA sequencing could provide important information of specific mRNAs stored in P-bodies of T and B lymphocytes at various stages of development. Furthermore, targeted mutagenesis of Pbody markers in T and B cells in vitro analyzed by various cell-based assays (e.g. proliferation, survival) and/or T or B-cell-specific knock-out in mice via Cre recombinase could help understand the specific functions of P-body subsets in T and B lymphocytes and their development. Even though I was able to confirm that RANTES mRNA is highly expressed in the cytoplasm of memory CD8+ T lymphocytes, I found that RANTES mRNA is not stored in P-bodies, but is diffusely distributed in the cytoplasm. Therefore, it is still an open question in immunology to understand how the translationally-repressed RANTES mRNA is stored. In my opinion, a good candidate for storage of RANTES mRNA is the stress-granule component fragile X mental retardation-related protein 1 (FXR1) that has previously been found to upregulate translation of stored mRNAs following a certain signal. 143, 146 Since FXR1 is a stress-granule protein, it is normally distributed diffusely in the cytoplasm, which is in agreement with localization of RANTES mRNA. In 179  preliminary experiments, I analyzed expression of FXR1 in naïve, activated, effector, and memory CD8+ T lymphocytes, but was not able to detect FXR1 in these cells either by flow cytometry or confocal microscopy, even following treatment with either 500 µM sodium arsenite (Redel-de Haen, Seelze, Germany #35000-1L-R) or 50 nM Pateamine A (from Dr. Jerry Pelletier, McGill University, Montreal, QC) that induce stress granule formation 176. However, I might have picked the wrong FXR1 Ab or a wrong stressgranule marker, and I believe that the role of stress-granule proteins in translational repression of RANTES mRNA is still a promising direction for investigation. For example, the stress-granule protein marker ZBP1 transports translationally-repressed mRNAs on F-actin filaments to the correct site of translation (reviewed in Refs. 193, 194). Therefore, co-localization analysis of FXR1, ZBP1, or another stress-granule marker with RANTES mRNA before and after treatment with sodium arsenite or Pateamine A could provide the answer whether RANTES mRNA is stored via translational repression by stress-granule proteins. Another important question that follows from the work presented in this thesis is whether IFN-γ mRNA stored in GWB+ RCK/p54+ P-bodies of memory CD8+ T lymphocytes is translated during the recall phase. I believe that a promising way to answer this question is to cross-link RNA-protein complexes before and after TCR engagement of memory CD8+ T lymphocytes, immunoprecipitate P-bodies, and compare levels of IFN-γ mRNA in cytoplasmic and P-body-associated fractions via qPCR. If resources allow, P-body CLIP followed by either Fluidigm analysis or mRNA sequencing could provide a wealth of information on kinds of mRNAs stored in P-bodies of T and B lymphocytes at various stages of development.  180  Assessment of P-bodies in memory CD8+ T lymphocytes generated in vitro is one of the limitations of this study. Even though I found that these memory cells closely resemble memory cells in vivo, the results I presented still need to be confirmed by assessing memory CD8+ T lymphocytes generated in vivo. In preliminary experiments, I (with help of Lisa Osborne) generated memory OT-I CD8+ T lymphocytes in vivo by infecting BoyJ (B6 background, CD45.1+) mice that received adoptively transferred naïve CD8+ T cells from OT-I (B6 background, CD45.2+) mice with OVA-expressing Listeria monocytogenes LM-OVA 195 (from Dr. Ninan Abraham, University of British Columbia) and re-isolating CD45.2+ cells as memory CD8+ T cells 8-12 weeks later. The number of memory CD8+ T lymphocytes generated using this method was not sufficient for a comprehensive analysis of P-bodies, but with the optimized protocols and increased number of mice, this method could be applied for evaluation of P-bodies in vivo. The original idea for the role of P-bodies in immune memory came from the reports on “memory-like” NK cells 8-10, which suggested that the current models for immune memory are incomplete, and thus additional mechanisms should exist. Therefore, addressing the role of P-bodies in these “memory-like” NK cells is an important future direction of this project. Finally, I found that GW182 localizes to the distal daughter cell during the first asymmetric division of naïve CD8+ T lymphocytes. Because IL-7Rα mRNA is also distributed to the distal daughter cell during this first asymmetric division 47, I believe it is an important question whether GW182 plays a role in the asymmetric distribution of IL-7Rα mRNA via direct interaction. This question could be addressed by both CLIPqPCR and confocal microscopy approaches. 181  6.4. Significance This study is the first to directly characterize P-bodies in T and B lymphocytes, as well as the potential role of P-bodies as effector mRNA storage sites, thus proposing a novel mechanism of immunological memory. A thorough understanding of immune memory could enhance the design of better vaccines or facilitate the development of novel vaccines for unmet medical needs. 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