Longterm impact of anti-CD20 mediated transient B cell depletion on memory B cells in patients with rheumatoid arthritis [Elektronische Ressource] = Langzeitveränderung der Gedächtnis-B-Zellen nach Anti-CD20-vermittelter B-Zelldepletion in Patienten mit rheumatoider Arthritis / submitted by Khalid Muhammad

Muhammad_K

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• Gesundheit

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2009
• Nombre de lectures: 8
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

LONGTERM IMPACT OF ANTI-CD20 MEDIATED TRANSIENT B CELL
DEPLETION ON MEMORY B CELLS IN PATIENTS WITH
RHEUMATOID ARTHRITIS


LANGZEITVERÄNDERUNG DER GEDÄCHTNIS B-ZELLEN NACH ANTI-CD20
VERMITTELTER B-ZELLDEPLETION IN PATIENTEN MIT RHEUMATOIDER
ARTHRITIS

Doctoral thesis for a doctoral degree
at the Graduate School of Life Sciences,
Julius-Maximilians-Universität Würzburg
Section: Infection and Immunity

Submitted by
Khalid Muhammad
Gujrat, Pakistan

Würzburg, 2009






























Submitted on: ……………………………………………………..……..
Office stamp

Members of the Promotionskomitee:

Chairperson: …………………………………………………

Primary Supervisor: Prof. Dr. Hans-Peter Tony

Supervisor (Second): Prof. Dr. Georg Krohne

Supervisor (Third): Dr. Ingolf Berberich


Date of Public Defence: …………………………………………….……

Date of receipt of Certificates: ...……………………………………….












To my parents, the dearest in my life!
To my wife Asifa and our lovely son Rayyan, with
love!























Contents


1 Introduction ............................................................................. 1
1.1 B-cell development in Human ................................................................................... 1
1.1.1 B cell ontogeny .......................................................................................................... 1
1.2 B cell activation..……………………………………………………………………4
1.3 Somatic hypermutation……………………………………………………………...5
1.3.1 Sites of somatic hypermutation...……………………………………………………5
1.3.2 Role of Transcription in somatic hypermutation ....................................................... 7
1.3.3 DNA double strand breaks and error-prone DNA repair .......................................... 7
1.6 Regulators of B cells………………………………………………………………..13
1.7 Functions of B cells ................................................................................... ………..15
1.8 Human memory B cells and marginal zone B (MZB) cells………………………..16

2 Autoimmunity .................................................................................................... 18
2.1 Rheumatoid arthritis………………………………………………………………..18
2.2 Pathophysiology of RA…………………………………………………………….20
2.2.1 Role of B cells in pathophysiology of RA ......................... ……………….………21
2.3 Therapeutic Targets in RA…………………………………………………………22
2.3.1 Biologics in RA ....................................................................................................... 23
2.4 B cell targeted therapies in RA ............................................................................... 25
2.5 Transient B cell depletion by anti-CD20 antibody Rituximab ………………….…26
Objectives of the project ............................................................... 28
Aims of study……………………………………………………………… 30

3 Patients,Materials and methods .......................................... 31
3.1 Patients .................................................................................................................... 31
3.2 Peripheral blood mononuclear cells (PBMCs) preparation………………………..32
3.3 Flow cytometry ....................................................................................................... 32
3.4 Cellular preparation for single cell sorting…………………………………………32
3.5 Single Cell sorting ................................................................................................... 33 3.5.1 Lower reaction mix ................................................................................................. 33
3.5.2 Sorting ..................................................................................................................... 34
3.5.3 Upper reaction mix .................................................................................................. 34
3.5.4 cDNA synthesis……………………………………………………………………34
3.6 Amplification of Ig-VH3 by nested PCR ................................................................ 35
3.6.1 Oligonucleotide Sequences ..................................................................................... 35
3.6.2 External Primers ...................................................................................................... 35
3.6.3 Internal Primers ....................................................................................................... 35
3.7 PCR amplification rounds ....................................................................................... 36
3.7.1 External amplification round………………………………………………………36
3.7.1 Internal amplification round...……………………………………………………..37
3.8 Primers used in Isotype studies……………………………………………………38
3.8.1 External Primers……………………………………………………………………38
3.8.2 Internal Primers ....................................................................................................... 39
3.9 PCR amplification rounds in Isotype studies .......................................................... 40
3.9.1 External PCR run .................................................................................................... 40
3.9.2 Internal/nested PCR run .......................................................................................... 41
3.10 Visulization of PCR product and DNA isolation from Agarose gel ....................... 42
3.11 Sequence Reaction .................................................................................................. 43
3.12 Purification of sequence reaction products ............................................................. 44
3.12.1 AutoSeq Column purification……………………………………………………...44
3.12.2 Isopropanol precipitation ......................................................................................... 44
3.13 An overview of Single B cells sorting .................................................................... 45
3.14 Sequencing .............................................................................................................. 46
3.15 Sequence analysis of Ig sequences .......................................................................... 46
3.16 Statistical analysis………………………………………………………………….48
3.17 Materials…………………………………………………………………………...49

4 Results .................................................................................... 51
4.1 Identification of Peripheral blood memory B cell subsets…………………………51
+
4.2 Peripheral blood CD27 memory B cells in patients with RA……………………..52
+4.3 CD21 surface expression of IgD memory B cells ................................................. 53
+ + +/-
4.4 Mutational pattern of IgD CD27 CD21 memory B cells .................................... 54
+ + +/-
4.5 Mutational pattern of IgD CD27 CD23 memory B cells 55 +4.6 A diversified IgR mutational pattern of IgD memory population .………………56
+
4.7 Rituximab mediated modulation of Ig-VH3 receptors of IgD memory B cells ....57
4.8 IgD+ memory B cells in SLE patient after rituximab ............................................. 63
4.9 Significant increase in CDR3 length after rituximab .............................................. 64
+4.10 V, D and J gene segments usage during repletion of IgD memory B cells ………66
4.11 Class-switched memory B cells during rituximab therapy ............................. ……67
4.12 V, D and J gene usage in class-switched memory B cells ………………………...69
4.13 Mutations in hotspot motif RGYW/WRCY during rituximab ……………………69
+
4.14 IgD memory B cells after allogeneic stem cell transplantation ………………….70
4.15 Class-switched memory B cells after ASCT ........................................................... 72
4.16 Autologous stem cell transplantation ………………………...................................73
4.17 Modulation of Ig-Isotypes during rituximab ……………………….......................74
5 Discussion .............................................................................. 76
6 References .............................................................................. 84
7 Summary…………………………………………………….97



Longterm impact of anti-CD20 mediated transient B cell depletion on memory B cells in patients with RA


1. Introduction

Despite their microscopic appearance that belies their functional heterogeneity and
development, lymphocytes known as T (thymus derived) and B cells (bursa or bone marrow
derived) have boosted the curiosity and challenged the experimental skills in many
disciplines. A simplified definition of B cells is a population of cells expressing clonally
diverse cell surface immunoglobulin (Ig) receptors which recognize specific antigenic
epitopes having their functional end point in antibody production by terminally differentiated
plasma cells (LeBien et al, 2008). The discovery and characterization of B cells occurred in
mid 1960s and early 1970s using animal models by