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Impaired Somatic Hypermutation and Increased Activation IMPAIRED SOMATIC HYPERMUTATION AND INCREASED ACTIVATION- INDUCED CYTIDINE DEAMINASE EXPRESSION DURING HIV-1 INFECTION by ELISABETH BOWERS B.S., University of Washington, Seattle, 2002 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Microbiology Program 2013 This thesis for the Doctor of Philosophy degree by Elisabeth Bowers has been approved for the Microbiology Program by Jerry Schaack, Chair Edward N. Janoff, Advisor Linda Van Dyk Larry Wysocki Cara Wilson Date 7/18/13 ii Bowers, Elisabeth (Ph.D., Microbiology) Impaired Somatic Hypermutation and Increased Activation-Induced Cytidine Deaminase Expression during HIV-1 Infection Thesis directed by Professor Edward N. Janoff ABSTRACT Background: HIV-1 infection is complicated by high rates of opportunistic infections against which specific antibodies contribute to immune defense. Antibody function depends on somatic hypermutation (SHM) of variable regions of immunoglobulin (Ig) heavy chain genes. SHM is mediated by a B cell-specific enzyme, activation-induced cytidine deaminse (AID). Methods: We characterized the frequency of SHM in expressed VH3-family IgD, IgM, IgA, and IgG mRNA immunoglobulin transcripts from control and HIV-1-infected patients using high-throughput pyrosequencing. We also compared AID mRNA expression by qRT-PCR, AID isoform expression by PCR, and the activation phenotype of B and T lymphocyte subsets among peripheral blood mononuclear cells (PBMC) from HIV-1-infected and control subjects pre- and post-stimulation. Results: VH3-IgM and VH3-IgA SHM frequencies did not differ between HIV-1- infected patients and controls. VH3-IgG SHM frequencies were significantly lower in HIV-1-infected patients as they were in another non-Ig AID target, Bcl6. VH3-IgD SHM frequencies were significantly higher in HIV-1-infected patients, however. Mutation iii patterns were comparable in both groups in all isotypes regardless of SHM frequency. AID mRNA expression was significantly higher in HIV-1-infected patients compared with controls. AID increased significantly post-stimulation in both groups, but the expression levels were lower among HIV-1-infected patients. At baseline, activation markers for B and T cells in multiple naïve and memory subsets were significantly higher in HIV-1-infected patients, but activation levels were not significantly different post- stimulation. AID expression correlated significantly with VH3-IgD SHM frequency and activation of T cells, but not with VH3-IgG SHM frequency, Bcl6 mutation frequency, B cell activation, or plasma HIV-1 RNA. AID isoform expression was comparable in both groups. Conclusions: B cells from HIV-1-infected patients show disparate SHM frequencies, especially amongst sequences known to control opportunistic infections which commonly cause morbidity during HIV-1 infection. Similar mutation patterns suggest differences in quantity, but not quality, of AID activity. Despite increased expression of AID mRNA and surface activation markers at baseline, B cells from HIV- 1-infected patients demonstrated a diminished capacity to upregulate AID mRNA in response to stimuli. These impairments may compromise humoral immune responses to both opportunistic infections and even to HIV-1 itself. The form and content of this abstract are approved. I recommend its publication. Approved: Edward N. Janoff iv ACKNOWLEDGEMENTS I would like to thank my family, especially my husband and my daughter, for all the sacrifices they have made and the support they have given me so that I could pursue my scientific goals. God has blessed me immensely with your support, encouragement and patience; so much more than I deserve. I would not have finished without your help. I would also like to thank all the friends that we have made during our time in Colorado who have also given us great encouragement and support when being so far from home. Many people contributed to the work presented in this thesis. The sequencing experiments could not have been possible without the valuable input from the Pollock lab, specifically Todd and Jill Castoe. Fantastic customer service was provided by Sergio Pereira at the Toronto TCAG Sequencing Core. Janoff lab and Frank lab current and former members, Diana Ir, Leah Feazel, and Emily Eshleman all contributed to generating and submitting sequences. Sequence analysis has been a huge challenge throughout this project, and the greatest contributor to my success in this arena is Dan Frank, who sacrificed a lot of time, hard-drive space, and RAM to analyze the massive amounts of data generated by this project. Initial efforts at Batch Analyzer coding by Abdul Rajib Bahar and Isaac Spitzer were also highly appreciated. Thank you also to Sam MaWhinney for thoughtful and tireless statistical analysis and to Tim Wright for patient recruitment at Denver Health Hospital. I am greatly appreciative also to the resident flow cytometry expert in the Janoff lab, Harsh Pratap, for endless hours spent v isolating PBMC and optimizing, running, and analyzing the flow cytometry data. Thanks also to Alison McMahon and Melissa Keays, former Janoff lab members, for flow expertise and effort. Other Janoff lab members have also been incredibly supportive and knowledgeable in helping me survive this experience with some sanity still intact; Jeremy Rahkola, Jana Palaia, Claire Gustafson, Jennna Achenbach, Rick Sullivan, and Jacinta Cooper. I would also like to thank my committee members Jerry Schaack, Linda van Dyk, Cara Wilson, and Larry Wysocki for all of the time that you have dedicated to this project and my advancement, for your patience, encouragement, and insightful suggestions. Finally, I would also like to thank my mentor, Edward Janoff, for all the time spent pushing me to work harder, think outside the box, and expand my scientific horizons. I am extremely appreciative of the time spent and the effort expended on my behalf. vi TABLE OF CONTENTS CHAPTER Page I. INTRODUCTION……………………………………………………………1 HIV-1………………………………………………………………………….1 Epidemiology……………………………………………………………...1 HIV-1 life cycle…………………………………………………….……..3 Pathogenesis……………………………………………………….………8 B cells………………………………………………………………..……….10 B cell development……………………………………………..………..10 Antibody structure……………………………………….………………18 Activation-induced cytidine deaminase…………………………...…..…21 Somatic hypermutation and class-switch recombination………….……..25 B cells and HIV-1………………………………………………….…….32 II. MATERIALS AND METHODS…………………………………………….35 Patient data…………………………………………………………..……….35 PBMC isolation and stimulation……………………………………………..36 Isolation…………………………………………………………………..36 Stimulation………………………………………………………….……36 Total immunoglobulins………………………………………………...…….36 mRNA isolation……………………………………………………..……….37 DNA isolation………………………………………………………………..37 vii cDNA generation………………………………………………………..…..37 Real-time PCR………………………………………………………………38 AID isoforms amplification and cloning…………………..………………...39 AID isoforms PCR………………………………………………..……..39 AID isoforms cloning………………………………………………...….40 Amplification, cloning, and sequencing of VH3-IgG genes………..………..41 PCR amplification of VH3-IgG genes……………………………………41 Cloning of VH3-IgG genes……………………………………………….43 Sequencing of VH3-IgG clones……………………………….………….43 VH3-IgG cloned sequence analysis…………………………………..………43 Determination of polymerase fidelity……………………………………43 VH3-IgG cloned sequence alignments and mutation calculations……….44 Mutation patterns analysis…………………………...…………………..44 CDR3 region analysis……………………………………..…………….45 VH3 454 PCR/High-throughput pyrosequencing…………………..………..45 st 1 round amplification…………………………………………………..45 nd 2 round amplification…………………………………………..………46 VH3 454-pyrosequencing analysis…………………………………….……..49 VhIGene program………………………………………………….……..49 Mutation patterns analysis………………………………………...……..50 CDR3 region analysis……………………………………………………50 Preliminary Bcl6 gene PCR, cloning, sequencing, and analysis…….………50 Preliminary Bcl6 PCR amplification……………………………………50 viii Cloning of Bcl6 genes………………………………………………..….52 Sequencing of Bcl6 clones…………………………………………..…..54 Bcl6 cloned sequence alignments and mutation calculations……...……54 Bcl6 454 PCR/High-throughput pyrosequencing……………….…………..54 1st round amplification…………………………………….…………….54 2nd round amplification………………………………………...………..55 Bcl6 454-pyrosequencing analysis……………………………….…………57 Flow cytometry………………………………………………………...……57 Statistical analysis……………………………………………………….…..58 III. THE SOMATIC HYPERMUTATION FREQUENCY OF VH3 FAMILY IMMUNOGLOBULIN GENES IS ALTERED IN HIV-1-INFECTED PATIENTS COMPARED WITH HEALTHY CONTROLS………………………………………………………….……..59 Introduction………………………………………………………….….59 Results………………………………………………………………..….62 VH somatic hypermutation frequency is reduced in VH3-IgG cloned samples from viremic HIV-1-infected patients but the mutation pattern is normal……………..………………………….…62 VH somatic hypermutation frequency is reduced in VH3-IgG 454- pyrosequenced genes from viremic HIV-1-infected patients but increased in VH3-IgD genes……………………………………….…82 Mutation frequency is reduced in Bcl6 genes in HIV-1-infected patients………………………………………………………...……115 Discussion………………………………………………………..……..123 Disparate SHM frequencies occur during HIV-1 infection……...…123 V(D)J recombination is normal during HIV-1-infection………...…124 Regulation of AID may be altered during HIV-1 infection……...…125 ix DNA repair pathways involved in SHM may be impaired during HIV-1 infection………………………………………………..……127 Antibody selection
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