THE ROLE OF B CELLS IN THE PATHOGENESIS OF ATHEROSCLEROSIS KHOO HAN BOON LAWRENCE (B.Sc (HONS.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MICROBIOLOGY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2013   i   DECLARATION I hereby declare that the thesis is my original work and it has been written by me in its entirety I have duly acknowledged all the sources of information which have been used in the thesis This thesis has also not been submitted for any degree in any university previously Khoo Han Boon Lawrence July 2013   ii   Acknowledgements First and foremost, this thesis is not possible without the generosity shown by my supervisor, Dr Veronique Angeli, who allowed me the freedom to define and pursue the scope of this project It was always rewarding and fruitful whenever we have a discussion no matter how short or long (or very long ones!) they could be Her guidance and quiet support are a pillar of strength and encouragement to rely on during the years Her financial support, both in overseas conferences and continuation of my candidature, has been a tremendous help in pursuit of my research interest For all these, I’ll like to express my deepest gratitude Thank you Next, I’ll like to thank my labmates who accompanied me along the journey of my graduate studies Thank you Fiona, for being a steadfast partner during mice harvest and your help in dissecting the aortas Thank you Jocelyn, for the helpful discussions To Angeline, Kim, Kar Wai, Serena, Sheau Ying and Michael, thank you for the help and support in lab matters I’ll also like to thank many friends I made in the Immunology Programme as well as in Singapore Immunology Network (SIgN) for the help they rendered in however big or small and not to mention the light-hearted moments! I’ll like to specially thank Dr Paul Hutchinson, Guo Hui and Fei Chuin for assistance in cell sorting experiments Also, special thanks to Dr Florent Ginhoux and Peter See for providing the CD11c-GFP-DTR mice Last but not least, I’ll like to dedicate this thesis to my family and express my deepest gratitude to my parents for their unwavering support in the pursuit of my graduate studies   iii   Table of Contents DECLARATION ………………………………………………… ii ACKNOWLEDGEMENTS …………………………………… … iii TABLE OF CONTENTS ………………………………………… iv ABSTRACT ………………………………… xv LIST OF TABLES xvii LIST OF FIGURES xvii LIST OF ABBREVIATIONS xxi Chapter 1: Introduction 1.1 Main Immunological Features in Atherosclerosis 1.2 Experimental Mice Models of Atherosclerosis 1.3 B cell subset populations 1.3.1 B1 cells 1.3.2 B2 cells 10 1.3.2.1 Follicular B cells 10 1.3.2.2 Marginal Zone B cells 11 1.4 Humoral Immunity in Atherosclerosis 12 1.4.1 Localization of B cells in Atherosclerosis 12 1.4.2 Association of oxLDL-specific antibodies with Atherosclerosis 14 1.4.3 Cloning of oxLDL-specific B cells 18   iv   1.4.4 B1 cell origin of oxLDL-specific monoclonal antibodies 19 1.4.5 Function of oxLDL-specific Antibodies 21 1.4.6 The role of B cells in Atherosclerosis: Pathogenic or protective? 22 1.5 Humoral B cell responses 24 1.5.1 Critical factors in differential antibody producing pathways 24 1.5.1.1 Antigen-BCR Interactions 25 1.5.1.2 Epstein-Barr virus-induced molecule (EBI2) 25 1.5.1.3 B cell responses master regulators 27 1.5.2 Extrafollicular responses 29 1.5.3 Germinal centres 34 1.5.4 Long-lived plasma cells 40 1.5.4.1 Migratory competency 44 1.5.4.2 Survival factors for plasma cells 47 1.5.4.3 Role of CD28 on longevity of plasma cells 50 1.5.4.4 Cellular sources of survival factors 51 1.6 Objectives 53   v   Chapter 2: Materials & Methods 2.1 Mice 55 2.1.1 Generation of mice chimera 55 2.1.2 Splenectomy 55 2.2 Immunization 56 2.3 Adoptive transfer 56 2.4 Ezetimibe treatment 56 2.5 BrdU and EdU administration 56 2.6 Plasma collection 57 2.7 Cell isolation from tissues 57 2.7.1 Isolation of cells from spleen and lymph nodes 57 2.7.2 Isolation of cells from bone marrow 58 2.7.3 Isolation of cells from peritoneal 58 2.8 Viability cell count 58 2.9 Flow cytometry 59 2.9.1 Cell surface staining 59 2.9.2 Intracellular staining 59 2.9.3 EdU staining for flow cytometry 60 2.9.4 5-bromodeoxyuridine (BrdU) staining 60   vi   2.9.5 Flow cytometry acquisition and analysis 61 2.9.6 Cell sorting 61 2.10 Enzyme-Linked Immunosorbent Assay (ELISA) 62 2.10.1 Quantification of total IgM and IgG 62 2.10.2 Measurement of anti-LDL and anti-oxLDL autoantibodies 62 2.10.3 Quantification of PC-specific antibodies 63 2.10.4 Measurement of OD and analysis 63 2.11 Enzyme-Linked Immunosorbent Spot (ELISpot) 64 2.11.1 Image acquisition and analysis 64 2.12 Cell culture 65 2.13 Immunofluorescence 66 2.13.1 Preparation of tissues for immunofluorescence staining 66 2.13.2 Immunofluorescence staining 66 2.13.2.1 Immunofluorescence staining with primary and/or secondary antibodies 66 2.13.2.2 Immunofluorescence staining with biotin-conjugated antibodies 67 2.13.2.3 Immunofluorescence staining with anti-mouse CD138 antibodies 67   vii   2.13.2.4 Revealing EdU+ cells in tissue sections 68 2.13.3 Image acquisition and analysis 68 2.14 Quantification of germinal center and extrafollicular responses 68 2.15 Quantification of lesion size 69 2.16 Oil Red-O staining 69 2.16.1 Image acquisition 69 2.17 Real-time Polymerase Chain Reaction (real-time PCR) 70 2.17.1 RNA Extraction 70 2.17.2 Reverse transcription 70 2.17.3 Primer Design 71 2.17.4 Real-time PCR Protocol 71 2.18 Mass spectroscopy analysis of samples 72 2.18.1 Extraction of sterols from tissue samples 72 2.18.2 High Performance Liquid Chromatography – Mass Spectrometry (HPLC-MS) method 72 2.19 Statistical Analysis 73   viii   Chapter 3: Results 3.1 Characterization of antibody responses in plasma of atherosclerotic experimental mice model, apoE-/- 75 3.1.1 Elevated total IgM and oxLDL-specific IgM autoantibodies in circulation with disease progression 75 3.1.2 No increase in PC-specific IgM autoantibodies in circulation 76 3.1.3 Summary 81 3.2 Comparative evaluation of humoral responses in secondary lymphoid organs – spleen and lymph nodes 82 3.2.1 Hypertrophy of lymph nodes, but not the spleen, in apoE-/mice 82 3.2.2 Evaluation of antibody-producing plasma cells in secondary lymphoid organs in apoE-/- mice 84 3.2.3 Evaluation of antibody producing pathways in secondary lymphoid organs in apoE-/- mice 86 3.2.3.1 Germinal center reactions increase significantly in the lymph nodes 86 3.2.3.2 Robust extrafollicular responses were observed in the spleen of apoE-/- mice 91 3.2.4 Evaluation of antibody responses in secondary lymphoid organs in apoE-/- mice 96   ix   3.2.5 Summary 100 3.3 Evaluation of antibody producing pathways in contributing total IgM and oxLDL-specific IgM autoantibodies in spleen of apoE-/- mice 101 3.3.1 Splenic GCs from apoE-/- mice were not defective in producing isotype-switched plasma cells 101 3.3.2 Plasmablasts in extrafollicular responses were IgM+ 104 3.3.3 Summary 108 3.4 Evaluation of molecular cues to direct extrafollicular responses in the spleen of apoE-/- mice 109 3.4.1 Increased ch25h mRNA expression in the spleen of apoE-/mice 109 3.4.2 Increased oxysterol in the spleen of apoE-/- mice 110 3.4.3 Summary 112 3.5 Antibody production of B1a cells in apoE-/- mice 113 3.5.1 B1a cells were not expanded in PEC of apoE-/- mice 113 3.5.2 Increased splenic B1a cells differentiation into IgM+ plasmablasts 114 3.5.3 Summary 118 3.6 Evaluation of the impact of splenic CD138+ ASCs on atherosclerosis 119   x   2.9 Flow cytometry 2.9.1 Cell surface staining Single cell suspension samples were maintained and stained with antibodies in fluorescence-activated cell sorting (FACS) buffer (0.5% bovine serum albumin (BSA)/2mM EDTA) Samples were routinely Fc receptor blocked with anti-CD16/32 to prevent false positive identification of cell population after antibodies staining Antibodies used are detailed in Appendix Samples were surface stained and appropriate isotype antibodies were included as controls After cell surface staining, the cells were fixed in 1% formalin for at least 20 minutes at 4oC Cells were resuspended with PBS in FACS tubes for flow cytometry acquisition 2.9.2 Intracellular staining After cell surface staining, samples were fixed and permeabilized for intracellular staining using the intracellular fixation and permeabilization buffer (eBioscience) as recommended Briefly, cells were fixed with the fixation buffer for 20 minutes at 4oC before intracellular antibodies staining in saponin-based permeabilization buffer for 20 minutes at 4oC All washes in between steps were with permeabilization buffer Cells were resuspended with PBS in FACS tubes for flow cytometry acquisition   59   2.9.3 EdU staining for flow cytometry To stain cells that incorporated 5-ethynyl-2’-deoxyuridine (EdU) for flow cytometry, Click-iT Edu flow cytometry assay kit (Invitrogen) was used according to manufacturer’s protocol Briefly, Click-iT fixative was used to fix the cells after surface antibodies staining and Click iT saponin-based permeabilization reagent was used to permeabilize the cells and as wash reagent Click-iT reaction cocktail composing of recommended amount of 1x Click-iT reaction buffer, copper sulphate, AF-647 azide and reaction buffer additive were added and incubated for 30 minutes at room temperature Antibodies that were phycoerthrin (PE)-conjugated and for intracellular staining purpose, were only used after revealing of EdU step as recommended 2.9.4 5-bromo-2’-deoxyuridine (BrdU) staining To stain cells that incorporated BrdU when they proliferate, BrdU flow kit (BD Pharmingen) was used as recommended in the manufacturer’s protocol Briefly, after cell surface antibodies staining, cells were fixed and permeabilized in BD Cytofix/Cytoperm Buffer and incubated 30mins on ice Cells were permeabilized further with BD Cytoperm Permeabilization Buffer Plus before re-fixing the cells with BD Cytofix/Cytoperm Buffer for minutes on ice Cells were treated with DNase to expose incorporated BrdU for hour at 37oC before staining with anti-BrdU antibodies for 20 minutes at room temperature   60   2.9.5 Flow cytometry acquisition and analysis Cell samples were acquired using either FACS Calibur on CellQuest software v5.2.1 (BD Bioscience) or Fortessa on FACSDiva software v6.1.3 (BD Bioscience) Data were analyzed on FlowJo software v8.8.4 (Tree Star) 2.9.6 Cell sorting After surface antibodies staining, cells were suspended in FACS buffer and sorted with MoFlo on Summit software (Beckman Coulter)   61   2.10 Enzyme-Linked Immunosorbent Assay (ELISA) 2.10.1 Quantification of total IgM and IgG To quantify the amount of total IgM and IgG present in the plasma, sandwiched ELISA assay was performed Briefly, Maxisorp 96-well plates (Nunc) were coated overnight with sheep anti-mouse Ig (Chemicon) at 5ug/ml in PBS and subsequently blocked with casein in PBS (Thermo Scientific) the following day hour at 37oC Mouse IgM and IgG (Invitrogen) were serially diluted in two-fold ranging from 500 to 7.8ng/ml and used as standard references for plasma samples Plasma samples were diluted at 1:100,000 and 1:1,000,000 with casein in PBS After overnight incubation at 4oC, horseradish peroxidase (HRP) conjugated goat anti-mouse IgM and IgG (Jackson Immuno) diluted with casein in PBS at 1:2500, were incubated 1hr 37oC for detection before addition of 2,2'-azino-bis(3-ethylbenzothiazoline-6sulphonic acid) (ABTS) substrate (Invitrogen) for color development The reaction was stopped with the addition of 0.1M citric acid All washes between the steps were performed with 0.05% PBS-Tween, followed by PBS 2.10.2 Measurement of anti-LDL and anti-oxLDL autoantibodies For anti-LDL and anti-oxLDL detection, LDL and oxLDL (BTI biotechnologies) were coated with 0.03mM EDTA in Tris-buffered saline (TBS) at 10ug/ml overnight The plates were subsequently blocked with casein in TBS (Thermo Scientific) Plasma were serially diluted in two-fold from 1:50 to 1:400 with casein in TBS and incubated overnight before incubation with HRP conjugated goat anti-mouse IgM and IgG diluted with casein in TBS at 1:2500 for hour at 37oC ABTS substrate was used for color   62   development and 0.1M citric acid was added to stop the reaction All washes were performed with 0.05% TBS-Tween followed by TBS 2.10.3 Quantification of PC-specific antibodies For PC detection, PC-BSA and BSA were coated with 0.03mM EDTA in TBS at 0.5µg/ml overnight The plates were blocked with casein in TBS for 1hr at 37oC Purified PC-specific IgM and IgG antibodies were serially diluted in two-fold from 200ng/ml to 3.125 ng/ml and used as standard references to plasma samples Both standards and samples added to the plate were incubated overnight at 4oC Following day, HRP conjugated goat anti-mouse IgM and IgG diluted with casein in TBS at 1:2500 for hour at 37oC ABTS substrate was used for color development was used and 0.1M citric acid was added to stop the reaction All washes were performed with 0.05% TBSTween, followed by TBS 2.10.4 Measurement of OD and analysis All plates were read at optical density (OD)415 using ELISA plate reader (Biorad model 680) with microplate Manager 5.2 software Standards were plotted in Excel v14.2.0 (Microsoft) and concentration of antibodies from each sample was calculated For PC-specific antibodies analysis, OD readings from plates coated with PC-BSA were subtracted from OD readings from BSA coated plate For measurement of titers of anti-LDL and anti-oxLDL antibodies, OD reading from each sample was subtracted from background OD reading   63   2.11 Enzyme-Linked Immunosorbent Spot (ELISpot) Goat anti-mouse IgM + IgG (20ug/ml) (Jackson Immuno), LDL or oxLDL (10ug/ml) were coated in nitrocellulose membrane plates (Millipore) overnight in PBS The plates were subsequently blocked with casein in PBS (Thermo Scientific) for 2-4 hours before overnight incubation with isolated cells in Roswell Park Memorial Institute (RPMI) (Sigma) supplemented with 10% (vol/vol) fetal bovine serum (FBS), penicillin-streptomycin and Lglutamate at 37oC For total IgM and total IgG analysis cells were seeded at 10,000 and 100,000 per well For anti-LDL and anti-oxLDL analysis, cells were seeded at 100,000 and 1,000,000 per well Prior to addition of secondary antibodies, all washes were performed with PBS between each step HRP conjugated goat anti-mouse IgM and IgG diluted with casein in PBS at 1:2500, were incubated hour at 37oC for detection before addition of 3-amino-9ethylcarbazole (AEC) substrate (BD Bioscience) Colour development was stopped when plates were rinse with running water All washes in between steps were performed with 0.05% PBS-Tween, followed by PBS Plates were dried in oven at 55oC before image acquisition 2.11.1 Image acquisition and analysis Plate images were captured and enumerated for spots using Immunospot Reader (CTL) with ImmunoSpot 5.0 Pro DC software   64   2.12 Cell culture Cell suspension isolated from spleen, lymph nodes, bone marrow and peritoneal cavity were cultured in complete RPMI (Sigma) supplemented with 10% (vol/vol) FBS, penicillin-streptomycin and L-glutamate Cells were seeded at million cells per well with 200µl of medium in 96-well round bottom plates for days Sorted GC B cells (B220+CD95+GL-7hi) from spleen were seeded at ~ 700,000 cells per well with 200µl of medium with IL-4 (50ng/ml) and antiCD40 (0.8µg/ml) in 96-well flat bottom plates for days Supernatant were collected for antibodies analysis after centrifuging at 1200rpm for minutes to remove cells   65   2.13 Immunofluorescence 2.13.1 Preparation of tissues for immunofluorescence staining Spleen and lymph node samples were freeze-embedded in OCT (Tissue Tekc) and kept in -80oC for storage For Edu-pulsed chase samples, tissues were fixed in 2% paraformaldehyde/ 30% sucrose at 4oC at least hours under gentle shaking and washed with 30% sucrose before embedding in OCT 6µm thick sections were made with cryostat machine (Leica) and mounted onto poly-lysine slides (Thermo Scientific) for storage at -20oC 2.13.2 Immunofluorescence staining 2.13.2.1 Immunofluorescence staining with primary and/or secondary antibodies To prepare slides for staining, they were dried under room temperature overnight To allow antibodies to be in placed after adding onto the samples, Dako pen (Dako) was used to create barrier around each sample and further drying of 30 minutes was required Slides with unfixed samples were fixed in ice-cold acetone for minutes Both acetone and paraformaldehyde fixed samples were blocked with 0.2% BSA for 10 minutes to minimize nonspecific binding of antibodies Normal mouse serum was used to block Fc receptors and as diluent for antibodies used Primary and secondary antibodies were incubated for at least 45 minutes at room temperature in humid chamber and kept in the dark Antibodies used for immunofluorescence staining are detailed in Appendix Subsequently, samples were stained with 4',6diamidino-2-phenylindole (DAPI) for minutes All washes in between steps were carried out times with PBS for minutes each Finally, samples were   66   mounted onto coverslips with fluorescent mounting media (Dako) to preserve fluorescence signals and sealed with nail varnish 2.13.2.2 Immunofluorescence staining with biotin-conjugated antibodies For use of biotinylated antibodies for detection, endogenous biotin present in tissue samples were blocked with avidin and biotin blocking kit (Vector Laboratories) as recommended by manufacturer’s protocol Briefly, avidin block was incubated with samples for 15 minutes before incubating biotin block for 15 minutes 2.13.2.3 Immunofluorescence staining with anti-mouse CD138 antibodies For tyramide-based amplification of signal for CD138 staining, 0.3% H2O2 was used to quench endogenous peroxidase activity for hour In addition, avidin/ biotin block were carried out as CD138-biotin antibodies were used Tyramide Signal Amplification (TSA)-biotin kit (Perkin Elmer) was used as recommended in manufacturer’s protocol to amplify signal Briefly, after CD138-biotin staining, streptavidin (SA)-HRP was added to samples and incubated for 30mins at room temperature Following, TSA-biotin diluted in amplification diluent was added for 10 minutes at room temperature After which, SA-Cy3 was added onto the samples to reveal CD138 staining before staining with other antibodies of interest on the tissue samples   67   2.13.2.4 Revealing EdU+ cells in tissue sections To reveal EdU+ cells in tissue sections after 12 hours pulsed-chase experiment, Click-iT EdU imaging kit (Invitrogen) was used according to manufacturer’s protocol Briefly, paraformaldehdye-fixed tissues were blocked with 0.2% BSA and treated with triton X-100 to permeabilize the cells in the tissue sections for 20 minutes at room temperature Click-iT reaction cocktail composing of recommended amount of 1x Click-iT reaction buffer, copper sulphate, Alexa Fluor azide and reaction buffer additive was added to the samples to stain Edu+ cells for 30 minutes at room temperature All washes in between steps were performed using PBS 2.13.3 Image acquisition and analysis Images of samples were captured using AxioCam Hrm camera (Zeiss) on fluorescent microscope (Zeiss) with AxioVision software v4.8.2.0 Where applicable, individual images of spleen and LNs were stitched to form the entire sample using MosiaX autostitch module in the software 2.14 Quantification of germinal center and extrafollicular responses PNA+ structure within the B cell follicles were identified to be GC reaction while the colocalization of CD11chi DCs with CD138+ plasmablasts at the bridging channel of follicle was identified as extrafollicular response site in the spleen Quantification of GC and extrafollicular responses in immunofluorescence staining was performed by dividing the number of GC and extrafollicular responses sites identified with the area of spleen analyzed in sample sections   68   using AxioVision software A total of 2-3 slides per spleen (at least 90µm between each slide used) were used and the calculated average value was representative of the extent of GC and extrafollicular responses taking place in the spleen A total of 2-3 slides per LN were used to enumerate the number of GCs present 2.15 Quantification of lesion size Quantification of lesion size of the aortic arch was made by measurement of the lesion area divided by the measurement of lumen area using AxioVision software A total of slides per sample were used and the calculated average value was representative of lesion size of these mice in the aortic arches 2.16 Oil Red-O staining Paraformaldehyde-fixed whole aorta was stained with Oil Red-O staining solution for 30-45 minutes for staining of lipids accumulated and washed in 60% 2-propanol until clear Whole aorta was stored in PBS until image acquisition 2.16.1 Image acquisition Images were captured using DP71 camera (Olympus) on stereomicroscope SZX16 (Olympus) with DP controller v3.3.1.292 software   69   2.17 Real-time Polymerase Chain Reaction (PCR) 2.17.1 RNA Extraction Tissue samples were stored in RNAlater (Qiagen) at -80oC until extraction Tissue samples were homogenized in Trizol reagent (Invitrogen) and RNA was extracted using NucleoSpin RNA II kit (Macherey-Nagel) according to manufacturer’s protocol Extracted mRNA concentration and purity was determined using Nanodrop 1000 Spectrophotometer (Thermo Scientific) 2.17.2 Reverse transcription Reverse transcription to synthesize cDNA was performed using Taqman Reverse Transcription kit (Applied Biosystem) on 2720 Thermal Cycler (Applied Biosystem) First, 200ng of each mRNA sample was linearized with 1.25µM of oligo-dT, 1.25µM of random hexamer and 500µM of dNTPs using the protocol as detailed in the following Step Linearization Stabilization Maintain Hold Hold Hold Time 5mins 2mins Infinite Temperature 65oC 4oC 4oC Table Linearization protocol Thereafter, final concentration of 1X Reverse Transcriptase buffer, 5.5mM of MgCl2, 0.4U/ µl of RNase inhibitor and 1.25U/ µl of multiscribe reverse transcriptase was added to each sample Elongation protocol detailed in the following was used   70   Step Incubation RT RT inactivation Hold Hold Hold Time 10mins 30mins 5mins Temperature 25oC 48oC 95oC Table Elongation protocol 2.17.3 Primer Design The design of primers for targeted genes was performed using GeneQuest v7.0.0 (DNA Star) Details of primers for respective target genes are detailed in Appendix 2.17.4 Real-time PCR Protocol Real-time PCR experiments were performed on 7500 Real Time PCR System (Applied Biosystems) with 7500 v2.0 software Each sample was composed of a total of 20µl volume composed of 10µl SYBR Green supermix with ROX (Bio-rad), 1µl of forward and reverse primers (1st Base), 7µl of Diethylpyrocarbonate (DEPC) water (Gibco) and 2µl of cDNA template Samples were maintained at 95oC for 10 minutes at the holding stage before amplification Forty-five cycles of amplification were performed with denaturation at 95oC for 15 seconds, annealing and extension at 60oC for 45 seconds as the cycling stage Data acquisition was collected at the annealing and extension phase Following the cycling stage, the continuous melt curve stage was performed as 95oC for 15 seconds, 60oC for minute, 95oC for 30 seconds and 60oC for 15 seconds   71   Expression levels were normalized to endogenous hypoxanthine phosphoribosyltransferase (HPRT1) with threshold cycle value set at 0.025 2.18 Mass spectroscopy analysis of samples The following protocol for measurement of oxysterols was adapted from the material and methods from previous studies (Hannedouche et al., 2011) 2.18.1 Extraction of sterols from tissue samples Spleen tissue organ samples were weighed and snap-frozen in liquid nitrogen before being pulverized with a CryoPrepTM (Covaris CP-02) The content was then transferred into glass micro reaction vials with internal standards (D6-7α, 25-OHC) and butylated hydroxytoluene (BHT) added before extraction Sterols were extracted in a Bligh/Dyer procedure The organic phase was separated and removed into a new glass vial The same procedure was applied to re-extract from the aqueous phase and precipitated protein All three extracts were subsequently pooled and dried under nitrogen stream at 40oC The residue was reconstituted with 100µl of 50% methanol (MeOH) for HPLC-MS analysis 2.18.2 High Performance Liquid Chromatography – Mass Spectrometry (HPLC-MS) method Ultra high performance liquid chromatography system (Nexra) coupled with a 5500QTrap mass spectrometer (AB SCIEX) was used An Acquity UPLCđ BEH C18 column (100 x 2.1mm x 1.7àm) with a pre-column (VanGuardTM, Acquity UPLC® BEH C18, x 2.1mm x 1.7µm) was applied at 55oC for chromatographic separation The mobile phase gradient program was used at a   72   flow rate of 400µl/min as follows (A: H2O + 5% MeOH + 0.1% FA; B: MeOH + 0.1% FA): 0.5 minutes, 35% B; minutes, 97% B; 6.5 minutes, 97% B 7α, 25-OHC was detected using multiple reaction monitoring transitions in the positive electrospray ionization mode as follows: 383 è 157, 159 and 365 è 157, 159 A MS/MS/MS transition 383 è 365 è 159 was used as a qualifier 2.19 Statistical Analysis Statistical analysis was performed using unpaired, two-tailed t-test in experiments with groups One-way ANOVA with Bonferroni’s multiple comparison post-test was performed with groups more than groups Twoway ANOVA with Bonferroni’s post-test was used for statistical analysis on LDL- and oxLDL-specific ELISA data All error bars represents S.E.M Prism software (Graphpad) was used for all statistical analysis   73   ... Marginal Zone B cells 11 1. 4 Humoral Immunity in Atherosclerosis 12 1. 4 .1 Localization of B cells in Atherosclerosis 12 1. 4.2 Association of oxLDL-specific antibodies with Atherosclerosis. .. further delineates the B1 population into B1 a (CD5+) and B1 b (CD5-) sub-populations B1 cell populations, unlike B2 cells, are not maintained through de novo influx of B1 cells to replenish the. .. 11 2 3.5 Antibody production of B1 a cells in apoE-/- mice 11 3 3.5 .1 B1 a cells were not expanded in PEC of apoE-/- mice 11 3 3.5.2 Increased splenic B1 a cells differentiation into IgM+