Aus dem Zentrum für Innere Medizin der Universität zu Köln
Klinik und Poliklinik für Innere Medizin I
Hämatologie und Onkologie
Direktor: Universitätsprofessor Dr. med. M. Hallek
Einfluss der Hypoxie auf den Immunphänotyp sowie auf die mRNA- und miRNA-
Expressionsprofile CD40-aktivierter B-Zellen (CD40Bs)
der Hohen Medizinischen Fakultät
der Universität zu Köln
ii
Gedruckt durch Hundt Druck GmbH, Zülpicher Str. 220, 50937 Köln
Gedruckt mit Genehmigung der Medizinischen Fakultät der Universität zu Köln 2016
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Dekan: Universitätsprofessor Dr. med. Dr. h.c. Th. Krieg
1. Berichterstatter: Professor Dr. med. Dr. rer. nat. M. S. von Bergwelt-Baildon
2. Berichterstatter: Universitätsprofessor Dr. rer. nat. R. J. Wiesner
Erklärung
Dritter und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe;
die aus fremden Quellen direkt oder indirekt übernommenen Gedanken sind als solche
kenntlich gemacht.
Bei der Auswahl und Auswertung des Materials sowie bei der Herstellung des
Manuskriptes habe ich keine Unterstützungsleistungen erhalten.
Weitere Personen waren an der geistigen Herstellung der vorliegenden Arbeit nicht
beteiligt. Insbesondere habe ich nicht die Hilfe einer Promotionsberaterin/eines
Promotionsberaters in Anspruch genommen. Dritte haben von mir weder unmittelbar
noch mittelbar geldwerte Leistungen für Arbeiten erhalten, die im Zusammenhang mit
dem Inhalt der vorgelegten Dissertationsschrift stehen.
Die Dissertationsschrift wurde von mir bisher weder im Inland noch im Ausland in
gleicher oder ähnlicher Form einer anderen Prüfungsbehörde vorgelegt.
Köln, 29. März 2015
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Die in dieser Arbeit beschriebenen Experimente sind von mir selbst ausgeführt und
ausgewertet worden.
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ACKNOWLEDGEMENTS
Many people have contributed to the completion of this thesis. I would like to express
my sincere appreciation towards Professor Michael von Bergwelt-Baildon for giving me
the opportunity to join his lab and explore the fascinating field of immunology. His
unwavering, contagious enthusiasm, optimism and vision have truly been a source of
inspiration time and again. I owe my deepest gratitude to my thesis advisor Dr.
Sebastian Theurich. This work would not have been possible if it were not for his
guidance, patience, knowledge and persistence during these years.
I would like to offer my special thanks to Dr. Alexander Shimabukuro-Vornhagen, Dr.
Lukas Frenzel and Malte Hülsemann for fruitful cooperations, discussions and valuable
advice. I am particularly grateful for the technical assistance provided by Anne Fiedler.
I must thank all of the members of the Bergwelt lab for their friendship and for creating
such an enjoyable atmosphere in the Haus 16 basement. Thanks in particular to
Shahram, Rieke, Joke, Tanja, Julia, Reinhild, Laura, Kerstin, Maria, Hans, Martin and
Sabrina. Every one of you has made available your support throughout the project. It
has been a pleasure to work with Professor Eisei Kondo. I benefited invaluably from his
vast knowledge and kindness.
I would like to thank the KölnFortune foundation for financially supporting this project.
No thanks to Beckman Coulter for their FACS acquisition and analysis software.
Finally, and most importantly, I would like to thank my family for their patience and
encouragement. I thank my parents and my sister for offering me unconditional and
unfailing support in all my pursuits. To them I dedicate this thesis.
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1.2 The CD40 costimulatory molecule .................................................................... 1
1.3 CD40-CD40L interaction and the B cell response ............................................ 2
1.4 CD40-CD40L interactions in the tumor microenvironment .............................. 3
1.5 CD40-activated B cells (CD40Bs) ..................................................................... 4
1.6 Hypoxia and the immune cell microenvironment .............................................. 6
1.7 The HIF transcription factor family ................................................................... 7
1.8 microRNA and B cell function .......................................................................... 9
1.9 Aim of this thesis ............................................................................................. 12
2 Material and Methods ..............................................................................................13
2.1 Cell Culture ...................................................................................................... 13
2.1.2 Buffers and Media .................................................................................... 14
2.1.3 Isolation of human PBMCs ...................................................................... 14
2.1.4 MACS CD19 positive selection ............................................................... 15
2.1.5 NIH 3T3 huCD40L cell culture ................................................................ 16
2.1.6 Human CD40-activated B cell cultures .................................................... 16
2.2 Fluorescence-activated cell sorting (FACS) .................................................... 19
2.2.1 FACS antibodies ....................................................................................... 19
2.3 Microarray analyses ......................................................................................... 20
2.3.2 Gene expression profiling ......................................................................... 22
2.3.3 microRNA expression profiling ............................................................... 26
viii
2.3.5 Statistics .................................................................................................... 31
3.1.3 altered expression of adhesion molecules CD62L and CD54 .................. 34
3.1.4 Shifts in B cell subpopulations under hypoxia ......................................... 36
3.2 Microarray analyses ......................................................................................... 38
3.2.2 Analysis of differential gene expression .................................................. 40
3.2.2.1 Day 1 differential gene expression .................................................... 40
3.2.2.2 Day 4 differential gene expression .................................................... 46
3.2.3 Analysis of miRNA expression ................................................................ 50
3.2.3.1 Day 1 differential miRNA expression ............................................... 50
3.2.3.2 Day 4 differential miRNA expression ............................................... 52
3.2.4 mRNA and miRNA matching analysis and functional enrichment ......... 54
4 Discussion ................................................................................................................56
of CD40B cells ............................................................................................................ 56
4.2 Differential regulation of cell adhesion molecules by hypoxia may influence B
cell interaction dynamics in SLO ................................................................................ 57
4.3 Shifts in B cell subpopulations under hypoxia ................................................ 58
4.4 Identification of hypoxia-responsive genes ..................................................... 60
5 Summary ..................................................................................................................66
6 Zusammenfassung ....................................................................................................67
7 References ................................................................................................................69
8 Anhang .....................................................................................................................80
APC antigen-presenting cell
BCL-2 transcriptional repressor B-cell lymphoma 2
BCR B-cell receptor
CXCL CXC chemokine ligand
DC dendritic cell
ddH2O double-distilled water
DMSO dimethylsulfoxide
EDTA ethylene-diamine tetraacetic acid
FasL Fas ligand
FITC fluorescein isothiocyanate
GC germinal center
gDNA genomic DNA
HEV high endothelial venules
IP1 buffer Inoculate PCR buffer 1
IVT in-vitro transcription
MEL extension and ligation master mix
MFI mean fluorescence intensity
MHC major histocompatibility complex
mRNA messenger ribonucleic acid
NIH 3T3 National Institutes of Health 3T3 murine fibroblast cells
NTP nucleoside triphosphate
p53 phosphoprotein 53
PB pacific blue
PBS phosphate buffered saline
PCR polymerase chain reaction
PMA phorbol-12-myristate-13-acetate
protein
UTP Uridine-5'-triphosphate
1
1.1 B CELLS AS ANTIGEN PRESENTING CELLS (APC)
As part of the adaptive immune system, B cells are arguably best known for their ability
to produce antibodies. However, increasing evidence suggests that B cells also
contribute to immunity by antibody-independent mechanisms. B cells can shift CD4 + T
cell differentiation between TH1 and TH2 cells by means of IL-4, IL-12 and IFN-γ
secretion. 54, 79
They may as well regulate immune responses directly by production of
cytokines such as IL-6, TNF-α or IL-10. 107
Upon activation, B cells can trigger cellular
immunity through processing and presentation of antigen to effector cells. In vitro,
peptide-pulsed activated B cells can present antigen to T cells in the context of MHC
class I and II molecules, inducing the activation of CD4 + helper and CD8
+ cytotoxic T
cells (CTL). 161
Antigen presentation by B cells has been proven to be indispensable for the induction of
normal T cell immunity. B cell depletion in mice from day of birth onwards resulted in
an abrogated T cell response when challenged with a specific antigen, a defect that
could be overcome by injecting B cells. 131, 132
B cell antigen presentation has also been
implicated in several pathologies. Mice bearing MHC class II-deficient B cells showed
prolonged survival in a cardiac allograft rejection model. 117
In another mouse model,
autoreactive antigen-presenting B cells were shown to be involved in the pathogenesis
of systemic lupus erythematosus (SLE) by inducing self-reactive T cells. 23
These
findings underline the B cell’s role as a potent antigen-presenting cell (APC). In line
with this hypothesis, B cell depletion with Rituximab (Mabthera), a chimeric
monoclonal anti-CD20 antibody, showed efficacy in treatment and prevention of
immune-mediated diseases such as acute graft-verus-host disease (GvHD). 72, 128
1.2 THE CD40 COSTIMULATORY MOLECULE
The costimulatory molecule CD40 is a member of the TNF receptor family (TNF
receptor superfamily member 5) which was initially found on B cells and is also
expressed on dendritic cells and monocytes. 8, 158
Its ligand CD40L, or CD154, belongs
to the same family of receptors and is primarily expressed on activated T cells. 158
Upon
engagement of CD40 by CD40L, adaptor proteins known as TNF-receptor associated
2
factors (TRAFs) 3 and 6 are recruited to the cytoplasmatic domain. This leads to the
activation of several downstream molecules including phosphatidylinositol-3 kinase
(PI3K), mitogen-activated protein kinase (MAPKs), phospholipase C (PLC), Jak and
STAT5 as well as NFkB via the canonical and non-canonical pathways. 12
The manifold downstream effects highlight the significance of CD40 signaling in B
cells, and indeed CD40 activation has been shown to be one of the main drivers of
germinal center (GC) formation, class switch recombination (CSR) and somatic
hypermutation (SHM), as described in the following sections. 36
Importantly, CD40
signaling mediates these functions in part by enhancing antigen presentation to T cells
and augmenting expression of costimulatory molecules.
1.3 CD40-CD40L INTERACTION AND THE B CELL RESPONSE
The B cell immune response is characterized by the formation of germinal centers
(GCs) in secondary lymphoid organs (SLOs). The events occurring during the course of
a thymus-dependent B cell response are discussed – albeit briefly – in this section.
At first, circulating naïve mature B cells enter primary lymphoid follicles in lymph
nodes and the spleen following a chemokine (CXCL13, C-X-C motif chemokine 13)
gradient. Activation of the B cell might occur due to crosslinking of the BCR by its
specific antigen, which is displayed on follicular dendritic cells (FDCs). Upon
activation, B cells upregulate CCR7 and migrate towards the border between the B cell
follicle and the T cell area. Here, B cells may encounter cognate helper T cells which
had been activated by the same antigen. The T cell recognizes the peptide:MHC II
complex as well as the costimulatory molecules CD80 and CD86 presented on the
surface of the activated B cell. In turn, in the T cell, these two stimulatory signals
trigger the increased expression of CD40 ligand (CD40L) on its surface, leading to
further stimulation of the B cell and subsequent, increased antigen presentation as well
as further upregulation of costimulatory molecules by the B cell. This stimulatory
cascade between cognate B and T cells eventually results in B cell proliferation and the
formation of a germinal center (GC).
In the GC, activated B cells expand while carrying out immunoglobulin class switch
recombination (CSR) and somatic hypermutation (SHM), thus promoting antibody class
3
centroblasts and centrocytes in the GC, eventually terminally differentiate into memory
or plasma B cells. As in the events leading to GC formation, interaction between CD40
on B cells and CD40 ligand (CD40L), expressed predominantly on follicular helper T
cells (TFH) in the GC, is crucial in GC maintenance and terminal cell differentiation as
well. 75
Underscoring these findings, it has been understood that individuals with
defective CD40-CD40L interaction suffer from hyper-IgM syndrome. Patients with this
condition fail to develop high affinity antibodies to thymus-dependent antigens and
immunoglobulin classes other than IgM. 105
1.4 CD40-CD40L INTERACTIONS IN THE TUMOR MICROENVIRONMENT
CD40-CD40L pathway activation is not limited to B and T cell interaction. Both
molecules are also expressed by different cells in the tumor microenvironment,
including tumor cells, endothelial cells, and tumor infiltrating lymphocytes (TIL). 86
Expression of CD40 and CD40L could be detected, amongst other neoplasms, in breast
cancers, 154
Some studies
suggest that expression of these molecules in tumors can drive cancer progression by
various mechanisms. For example, it has been shown that CD40 ligation can induce
vascular endothelial growth factor (VEGF) secretion in CLL cells, leading to enhanced
cell survival. 37
Coexpression and autocrine stimulation of the CD40/CD40L pathway
can constitutively activate NFkB signaling, as Pham et al. have demonstrated in
aggressive B cell lymphoma. 123
CD40 ligand may also confer resistance to
chemotherapeutic agents, which has been verified in vitro with gastric cancer, non-
Hogdkin lymphoma (NHL) and breast carcinoma cell lines. 163
In line with these
findings, correlation with clinical data has shown a clear association between soluble
CD40L (sCD40L) levels and tumor progression and metastasis in patients with
carcinoma of the lung and gastric cancer. 98, 133
On the other hand, CD40 activation of cells in the tumor microenvironment can also
exert antineoplastic effects. Firstly, CD40L can induce activation-induced cell death by
directly binding to CD40 on cancerous cells, as seen with melanoma, breast cancer and
glioma cells. 32, 154, 162
Secondly, CD40/CD40L interaction enhances the immune
response against the tumor by activating antigen-presenting cells within the tumor
4
microenvironment. For example, CD40 activation of dendritic cells can offset the effect
of tumor-derived immunosuppressive cytokines and can rescue these cells from
apoptosis. 124
Administration of stimulating anti-CD40 antibodies has been shown to
support immune control over tumors by macrophages, another type of antigen-
presenting cell (APC). 18, 106
As B cells represent a sizable fraction of the tumor immune infiltrate, 143
it can be
hypothesized that CD40 activation of this population yields a biologically relevant anti-
tumor effect as well. Interestingly, a study by Jackaman et al. could show that the anti-
tumor response observed after local application of an agonistic anti-CD40 antibody into
the tumor was dependent on the presence of B cells and independent of other APCs. 67
These findings underline the role of the CD40 and CD40L molecules as potent
immunostimulators not only in the physiological immune response, but also in
neoplastic tissues. However, to date, there is no data on how the environmental cues
modulate B cell activation within the tumor microenvironment.
1.5 CD40-ACTIVATED B CELLS (CD40BS)
Analogous to the events in SLOs the GCs, B cells can be stimulated via the CD40
pathway in vitro rendering a model for T cell dependent B cell activation. In the early
1990s, it was demonstrated that CD40 and IL-4 stimulation of primary human B cells
suffice to generate long-term, clonally expanding B cell lines, dubbed CD40B cell
cultures. 7 Later, it was shown that CD40Bs, generated from a small amount (50-100 cc)
of purified peripheral blood, are efficient antigen-presenting cells that were able to
induce proliferation of allogenic T cells. Their stimulatory capacity, in combination
with the ready availability and rapid expansion in CD40B cultures, raised interest in
CD40Bs as possible antigen-presenting cells (APCs) used in ex-vivo generation of
autologous specific T cells for adoptive T cell transfer. 138
This idea was explored in
several studies showing that CD40Bs can indeed present antigen and induce
proliferation of antigen-specific T cells from naïve T or antigen-experienced T cells.
Antigen could be delivered by peptide or protein pulse, addition of tumor lysate to the
culture, RNA transfection or retroviral transduction. 2, 82, 92, 161
CD40B cells generated from human peripheral blood mononuclear cells (PBMCs)
express high levels of MHC class I and II molecules, adhesion markers such as CD11a,
5
CD54 and CD58, as well as costimulatory molecules CD80 and CD86. 138, 169
Expression
of these proteins was similar when measured in CD40Bs compared to dendritic cells
matured with either CD40L or TNF-α. 138
In mixed lymphocyte reactions (MLRs), allogenic stimulatory capacity of CD40Bs was
shown to be inferior to DCs when solely the number of proliferating T cells per antigen-
presenting cell (APC) was considered, meaning that higher numbers of CD40Bs were
necessary to generate the same amount of T cells induced in DC-based stimulation
systems. 138
This discrepancy is explained by the difference in cell surface area, as DCs
possess long cellular processes enabling them to stimulate multiple T cells at once.
However, this advantage can be offset by the scalability of CD40B cell cultures: They
are less difficult to establish when compared with DC cultures and can be expanded
over an extended period of time, allowing for the rapid expansion of virtually unlimited
numbers of cells. A study by Wiesner et al. observed continuous CD40B cell
proliferation over more than 1650 days, representing 370 population doublings,
suggesting that these B cells have been conditionally immortalized. Importantly,
CD40Bs sustained their phenotype and expressed stable levels of stimulatory
molecules. 169
Furthermore, compared with DCs, CD40Bs can induce T cells that
express more IFN-γ, implicating not only a quantitative, but also a qualitative difference
between DCs and CD40Bs regarding T cell stimulation signals. 138
Next to the adoptive transfer of in vitro CD40B-primed specific T cells, induction of
immunity in vivo by administrating antigen-presenting CD40Bs as cellular adjuvants
has been suggested as a novel immunotherapeutic approach. For CD40Bs to mount an
immune response, they must relocate to tissues where they are likely to encounter and
attract cognate T cells. It has been demonstrated that CD40Bs express CD62L,
CCR7/CXCR4 and LFA-1, the adhesion and receptor molecules necessary for migration
to secondary lymphoid organs (SLOs). 160
In addition, expression of T cell-attracting
cytokines, such as CCL17 (TARC), CCL5 (RANTES), CCL22 (MDC), and CXCL10
(IP-10) could be observed. 160
In vivo homing of CD40Bs to the B-zone/T-zone
boundary of SLOs and chemoattraction of T cells could be shown in a mouse model. 77
Finally, the ability to prime antigen-specific immune responses in vivo has also been
verified in different settings, including a mouse melanoma model and a trial with
privately owned dogs suffering from non-Hodgkin’s Lymphoma. 51, 110, 145
6
Owing to the simplicity and efficacy of this established model, the CD40B culture
system is considered a promising source of antigen-presenting cells for use in
immunotherapy and represents a suitable in vitro model for activated B cells.
1.6 HYPOXIA AND THE IMMUNE CELL MICROENVIRONMENT
In vivo, immune cells are exposed to various environments as they differentiate and
migrate to different tissues. 21
Specifically, antigen presentation and immunomodulation
by B cells often occur in extreme situations, for example in inflammatory areas or in
tumors. Among other features, like increased acidity, 74
these environments are
characterized by tissue hypoxia.
Physiologically, oxygen levels decline from 21 kPa in the inspired room air, to lung
alveoli at 13 kPa, to 5 kPa in central veins. Once oxygen has entered the tissues, levels
decrease proportionally with increasing distance to the next capillary. In sites of high
metabolic turnover and subsequent increased oxygen demand, for example lymphoid
organs or neoplastic tumors, oxygen tensions can be dramatically low. Measurements in
mouse thymus revealed partial pressures of ~1kPa. 15
Using microelectrodes, partial
pressures in spleens of anaesthetized, live mice were shown to vary from ~5 kPa near
the splenic arteries to ~0.5 kPa in the periphery. 21
The same study could demonstrate
that although cytotoxic T cell (CTL) lethal hit delivery, i.e. perforin or Fas/FasL-
mediated cell lysis, was not influenced by hypoxia (pO2 2.5 kPa), development of a
more sustained and potent CTL population was observed when cultures were kept in
hypoxia. Furthermore, the authors described a shift in cytokine production from
proinflammatory cytokines IL-2 and IFN-γ to hypoxia-sensitive cytokines such as
VEGF. 21
Chronic hypoxia down-regulated the expression of the KV1.3 potassium
channel in human T cells as well as in the Jurkat T cell line. 29
This channel plays an
important role in maintaining the resting membrane potential in T lymphocytes and
mediates the influx of calcium upon T cell activation. Inhibition of KV1.3 led to
membrane depolarization and a decreased activation response. Of note, mRNA levels
were not altered in hypoxic cells, suggesting a posttranscriptional regulation
mechanism. 29
Contrary to the findings in T cells, hypoxia was found to enhance production of
proinflammatory cytokines TNF-α and IL-1 in macrophages. 43, 178
Yet, is has been
demonstrated that phagocytic activity was reduced in macrophages exposed to hypoxia,
which correlates with other findings of decreased ATP stores and impaired formation of
reactive oxygen species. 96
Another study showed that during acute lung injury, tissue
hypoxia prevented an overacting immune reaction in an adenosine and adenosine
receptor-mediated, lung-protective pathway. 153
Anti-inflammatory protection could be
Thus, hypoxia may modulate both proinflammatory and inhibitory mechanisms during
the immune response, depending on cell type and specific environment.
In spite of the aforementioned, in vitro experiments are usually carried out under
standard conditions with 21% oxygen and 5% carbon dioxide, which must be
considered a “hyperoxic” environment in physiological terms. Observations made in
this experimental setting might not reflect immunologic principles that are valid in vivo.
1.7 THE HIF TRANSCRIPTION FACTOR FAMILY
Cells possess mechanisms to actively adapt to low oxygen levels. On the gene
expression level, this hypoxic response is mediated by the family of hypoxia-inducible
factor (HIF) transcription factors. HIF-1 was initially identified as a transcriptional
enhancer of the erythropoietin (EPO) gene in human liver and kidney cells. 140
Subsequently, it was established that HIF-mediated transcription is common in many
mammalian cells and is responsible for hypoxia-induced expression of a plethora of
genes. 165
The HIF transcription factor is heterodimer consisting of the HIF-1β subunit (also
known as aryl hydrocarbon receptor nuclear translocator, ARNT), which is
constitutively expressed, and either one of the hypoxia-sensitive isoforms HIF-1α or
HIF-2α. The HIF alpha subunits contain a basic helix-loop-helix domain (bHLH) for
DNA binding and a Per-Arnt-Sim (PAS) domain facilitating protein dimerization. 57, 69
More recently, the existence of a third alpha subunit, HIF-3α, has been reported. 53
This
isoform lacks transcriptional activity and is thought to negatively regulate hypoxia-
induced gene expression, counteracting the other HIF isoforms. 109
Response to oxygen levels is mediated exclusively by the HIF-alpha subunits. The
stability of these proteins is regulated posttranscriptionally by oxygen- and iron-
8
dependent prolyl hydroxylases (PHDs) in the cytosol. In the presence of oxygen, PHDs
hydroxylate the HIF-alpha subunits. Hydroxylation is a key enzymatic step required for
the subsequent ubiquitinylation of the protein in a von Hippel-Lindau (vHL) protein-
dependent process. Ultimately, this leads to the proteasomal degradation of the HIF-
alpha subunits and suppression of HIF-induced gene expression. In hypoxic conditions,
failure of PHDs to hydroxylate HIF-alpha averts its degradation and leads to its
accumulation in the cytosol. After translocation into the nucleus, the HIF-alpha subunit
can dimerize with HIF-1β and bind additional coactivators such as P300. 142
The
complex can then initiate the transcription of genes whose promoters contain hypoxia
responsive elements (HRE). The consensus sequence for HREs is 5'-[A/G]CGTG-3'. 141
Apart from hypoxia-dependent stabilization, HIF activity can also be induced in
response to other extracellular cues unrelated to oxygen levels. LPS from gram-negative
bacteria induced transcription of HIF-1α in macrophages under normoxia, a process
promoted by activation of NFkB (nuclear factor kappa-light-chain-enhancer of activated
B cells). 14, 130
In T cells, ligation of the T cell receptor led to a PI3K/mTOR-dependent
accumulation of HIF-1α, which also promoted differentiation towards proinflammatory
T helper 17 (Th17) cells. 62, 114
Thus, HIF may play a role as an integrator of
environmental (hypoxia) and immune receptor-mediated (TLR, TCR) signals in
different immune cells during the inflammatory response.
Of all known isoforms, HIF-1α is the one most extensively studied and was found to be
widely expressed in many different tissues. In cells of the innate and adaptive immune
system, HIF-1α expression has been observed in neutrophils, macrophages, dendritic
cells and T and B lymphocytes. 31, 68, 90, 164
Though HIF-2α expression has not been
investigated as comprehensively, its expression could be shown in macrophages and
CTL. 35, 64
In non-immune cells, it has been established that besides a set of shared target
genes, both isoforms induce the expression of distinct targets. 58, 103
The stimuli inducing
specific activation of either HIF-1α or HIF-2α still remain mostly unclear.
To date, there is only limited data on the functional impact of HIF-1α on B cells. HIF-
1α knock-out studies initially proved to be challenging as HIF-1α has a crucial role in
embryonic development, leading to lethality of HIF-deficient mice. This obstacle was
addressed with the recombination-activating gene 2 (RAG2)-deficient blastocyst
complementation system. 80
RAG2-deficient mice are unable to produce T or B cells.
Blastocysts of RAG2-deficient mice were complemented with HIF-1α knock-out
9
embryonic stem (ES) cells, which gave rise to HIF-1α-deficient B and T cells in the
resulting chimera. Interestingly, the effects of HIF-1α deficiency were lineage-specific
and affected the development of B cells only. An abnormally large proportion of
peritoneal B cells in these mice showed a B1 cell-like phenotype, which was associated
with markedly increased levels of IgM and IgG ds-DNA autoantibodies, rheumatoid
factor as well as proteinuria. 80
Taken together, these in vivo experiments suggested that
HIF-1α might represent a negative regulator in B cells. The same study also showed
perturbed maturation of conventional B2 cells at the pre-B cell level. Later
investigations revealed that the transition from the pro-B to the pre-B cell stage was
largely dependent on the glycolytic pathway. Deficiency of HIF-induced glucose
transporter (GLUT) and phosphofructokinase/fructosebisphosphatase (PFKBP)
transcription led to energy shortage and cell death of pro-B cells, resulting in decreased
production of viable, immature B cells in the bone marrow. 81
Loss of HIF-1α abolished
hypoxia-induced growth arrest in splenic B cells. 45
The effect of hypobaric hypoxia on antibody production was examined in an
immunization study. 11
N. meningitides polysaccharides in high altitudes (4900 m) were not significantly
different from titers of subjects immunized at sea level. Spectrotypic analysis also failed
to detect qualitative differences in the antibody repertoires.
In summary, although some literature on the role of HIF in B cell development and
function is available, there currently is a paucity of data concerning the effects of HIF-
mediated gene expression on antigen presentation in B cells.
1.8 MICRORNA AND B CELL FUNCTION
MicroRNAs (miRNAs) belong to a class of non-coding RNA which was first
discovered in Caenorhabditis elegans in 1993. 95, 170
miRNAs have since been
recognized as key players of post-transcriptional gene regulation conserved in animals,
plants and even select viruses. 9 They can bind specific target messenger RNAs, leading
to their degradation which results in subsequent silencing of the gene. Estimates have
predicted that >60% of human protein-coding genes are directly regulated by
miRNAs. 40
The current official miRNA databse, miRbase 21, includes 2588 mature
human miRNA sequences. 38
10
In the classical model of miRNA biogenesis, miRNA arise from long, primary RNA
(pri-miRNA) that have been transcribed by RNA polymerase II. 179
These transcripts are
polyadenylated and capped. Further nuclear processing is carried out by the so-called
microprocessor complex, which consists of the endonuclease Drosha and its cofactor
DiGeorge Syndrome Critical Region protein 8 (DGCP8). The microprocessor complex
cuts both strands of the hairpin-shaped pri-miRNA loop. In the cytoplasm, the resulting
double-stranded, 60-70 nucleotide (nt) long precursor miRNA (pre-miRNA) is cleaved
by Dicer, another endonuclease, yielding the 20-22 nt long mature miRNA and its
complementary strand, denoted miRNA*. The mature miRNA strand is then unwrapped
and incorporated into the RNA-induced silencing complex (RISC), a complex
containing several proteins including the Argonaute (Ago) protein family. mRNA
transcripts bearing a nucleotide sequence in their 3’ untranslated region (3’UTR) which
is complementary to the sequence of the miRNA are bound by the miRNA-containing
RISC complex. Depending on the degree of complementarity between miRNA and
targeted mRNA, the mRNA transcript is either directly degraded by the RISC-complex
or translation of the transcript is repressed, leading to decreased expression of the gene.
However, in recent studies, a translation-enhancing function of miRNAs has been
identified, demonstrating that miRNAs may play different roles depending on the
cellular context. 159
miRNAs have been shown to participate in B cell development and function. When
miRNA maturation was suppressed in B lineage cells by specific Dicer deletion using a
CD19-induced Cre recombinase system, transition of B cells from the pro-B to pre-B
cell stage was blocked. 85
Gene expression profiling revealed overexpression of genes
targeted by the miR-17-92 family, consisting of miR-17, -19a, -19b, -20a and -92.
Targets included BCL2L11 (Bcl-2-like protein 11), which codes for pro-apoptotic
protein Bim, and B cell development could be partially rescued by suppression of Bim
overexpression. 85
Ablation of miRNA generation at a later stage of B cell development
was accomplished using mouse models in which Dicer deletion was conditionally
linked to AICDA gene expression. This allowed for a study by Xu et al. which
investigated miRNAs in terminal B cell differentiation, i.e. in activated B cells. 174
After
challenge with a T cell dependent antigen, serum analysis showed that although antigen-
specific IgM levels were comparable, IgG was significantly reduced in Dicer-deficient
mice compared to wild type controls. Antigen-specific memory B cells and plasma cells
were absent in the spleen and bone marrow of mutants. The formation of germinal
11
centers, the histological correlate of B cell affinity maturation and differentiation, was
impaired in these mice as well. These findings suggest that successful production of
high-affinity, class switched antibodies and the generation of antigen-specific effector B
cells are, at least in part, Dicer-dependent and are under the control of miRNAs. 174
Belver et al. could demonstrate that Dicer deletion in transitional B cells leads to
skewed terminal B cell subsets, favoring the generation of marginal zone (MZ) B cells
at the expense of a diminished follicular (FO) B cell subset. 10
Follicular B cells are the
protagonists of T cell-dependent B cell responses required for the generation of highly
diverse antibodies. Marginal zone B cells, located in the spleen marginal sinus, provide
rapid, T cell-independent humoral responses and are associated with the production of
self-reactive antibodies. 99
increased ds-DNA and cardiolipin autoantibodies. After further analysis miR-185 was
identified to play a central role in the FO versus MZ B cell fate decision by targeting
burton tyrosine kinase (BTK). 10
Modulation of BCR signaling by means of BTK
downregulation appears to be crucial for a normal differentiation pattern of peripheral B
cell subsets.
To date, no miRNA has been reported to directly participate in antigen presentation by
B cells. 33
Although antibody-independent functions of activated B cells have gained scientific
interest in the past years, there is a scarcity of knowledge on how these cells respond to
environmental challenges. Specifically, the influence of hypoxia, a physiological
condition encountered regularly by B cells, has so far remained largely unexplored.
Therefore, the primary objective of this thesis was to identify the effects of hypoxia on
the antigen presentation function of CD40-activated B cells (CD40Bs). To this end, this
project focused on three aspects of the hypoxia-induced “reactome”:
1) Immune phenotyping: Evaluation of differences in B cell surface marker
expression under hypoxic conditions using 10 color flow cytometry.
2) Gene expression analysis: Identification of differentially regulated genes in
normoxic and hypoxic CD40B cells with Illumina BeadArrays. This was
followed by functional enrichment to detect gene networks that are involved in
B cell antigen presentation.
miRNAs using Illumina BeadArrays. Subsequent integration with the gene
expression data was to be performed in silico to detect potential interactions that
might represent a regulatory mechanism in hypoxia-mediated differential gene
expression.
This relatively broad approach was intended as a first step towards understanding how
activated B cells possibly behave under in vivo conditions. Future investigations might
build on the data generated in this study to define the precise molecular mechanisms
involved.
13
All cell culture operations were conducted under sterile hoods (Heraeus, Germany)
when required. 1000, 200, 10 and 2 µl pipette tips (Sarstedt), 50 ml and 15 ml tubes
(Falcon), 1.5 ml Eppendorf tubes, and glass bottles were autoclaved before use.
Surfaces in the cell culture facility were treated with ethanol 70% before performing
experiments.
Cells were kept in an incubator (Heraeus) at 37°C and a constant CO2 fraction of 5%.
For experiments described as conducted under normoxic conditions, cells were cultured
in ambient room oxygen (21% FiO2, ca. 1025 hPa in Cologne, 6 m above NN).
Hypoxia experiments were performed in a custom-made airtight box made of acrylic
glass (courtesy of Prof. Noegel and O. Feierabend, Institute for Biochemistry, Cologne
University Hospital). A gas mixture composed of 95% nitrogen (N2) and 5% CO2 was
introduced into the box, displacing the oxygen. During the exchange of the gas, the
remaining oxygen content in the displaced air was measured with an oxymeter
(GOX100, Greisinger) at the gas outlet. Introduction of the N2 gas mixture was
continued until the oxygen fraction in the displaced air reached 0.8 % percent. The gas
outlet and inlet were then sealed and the box was placed into the incubator.
Light microscopy to monitor cell cultures was performed under an inverse phase
contrast microscope (Carl Zeiss). A Neumann hemocytometer was used to determine
cell concentrations of cell suspensions (Optik Labor) after dilution (1:10) and staining
with trypan blue solution to distinguish between viable and non-viable cells.
Centrifugation of 50 and 15 ml tubes as well as FACS tubes was carried out in a
Megafuge R1.0 (Heraeus) using rotor #3524. Eppendorf tubes were centrifuged in a
benchtop device (Heraeus).
2.1.3 ISOLATION OF HUMAN PBMCS
Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density
gradient centrifugation of buffy coats obtained from healthy donor blood. A buffy coat
is the remaining blood cell fraction after separation of erythrocytes and thrombocytes
from whole donor blood. It contains predominantly leukocytes at a very high
concentration. The buffy coats for all experiments were collected from the blood bank
of the University Hospital of Cologne. All donors gave their consent prior to donation.
From 50 ml of total buffy coat volume, 25 ml were transferred to two 50 ml Falcon
tubes. The buffy coat was diluted by adding 25 ml of PBS (1:2 dilution) and mixed by
inverting the tube 2-3 times. The diluted cell suspension was then transferred to 3 fresh
50 ml Falcon tubes containing 15 ml Ficoll-Paque (Pancoll human, density 1.077 g/ml,
PAN Biotech), gently layering the suspension on top of the Ficoll so that 2 separate
layers form without mixing. The tubes were then centrifuged for 20 minutes at 1080 g
(2400 rpm) without brakes at room temperature. After centrifugation, the leukocyte-rich
interphase was collected and transferred to a single fresh 50 ml Falcon tube. PBS was
Table 1 Buffers and media
CD40B Medium
+ 10% FCS PAA
+ 1 % Pen/Strep
10x Dulbecco’s PBS (DPBS) PAA, Cat. H15-011
diluted to 1x PBS with ddH2O pH adjusted to 7.2
sterilized by autoclavation
+ 0,5 % BSA Sigma Aldrich
15
added to a total of 50 ml and the tubes were centrifuged for 7 minutes at 1300 rpm, after
which the supernatant was discarded. The washing procedure was repeated once. After
centrifugation the cell pellet was resuspended in 20 ml culture medium. Cell
concentration and total cell count were subsequently determined.
2.1.4 MACS CD19 POSITIVE SELECTION
CD19-positive B cells were isolated by microbead positive selection using magnetic-
activated cell sorting (MACS, Miltenyi Biotec). With this method, cells are incubated
with antibodies against a surface antigen expressed on the cell population of interest.
The antibodies are covalently linked to paramagnetic particles (iron beads). After
binding, the suspension is transferred to a column which is exposed to a strong magnetic
field. Cells that do not express the antigen and thus are not labeled with an antibody
flow through the column, whereas the cells of interest bearing the antigen are
immobilized within the column due to magnetic forces. These cells can finally be
washed out after removing the column from the magnetic field.
In this study, CD19 positive B cells from 6 buffy coats were isolated by MACS positive
selection for one experiment. Cells were washed in cold PBS once, followed by
centrifugation (300 g for 10 minutes). The supernatant was discarded and the cell pellet
was resuspended in 0.08 ml of cold MACS buffer per 10 7 cells. CD19 microbeads
(Miltenyi) were added at 0.01 ml per 10 7 cells. Cells were then incubated at 4 °C for 15
minutes with gentle agitation. After labeling, cells were washed with 50 ml cold MACS
buffer and centrifuged for 10 minutes at 300 g. The supernatant was discarded and the
cell pellet was resuspended in 0.5 ml MACS buffer per 10 8 cells. The suspension was
then kept cold on ice. Miltenyi LS columns (cat. 130-042-401) were placed into
MidiMACS separators containing a strong magnet (Miltenyi, cat. 130-042-302). A 200
µm filter (Miltenyi) was placed on the top orifice of the column and both filter and
column were flushed once with 3 ml of MACS buffer. The cell suspension containing
labeled B cells was then applied to the filter and the cells were allowed to pass through
the filter and the column. Filter and column were subsequently rinsed 4 times with 3 ml
of MACS buffer to wash out CD19-negative cells. After removing the column from the
MidiMACS separator, CD19-positive B cells were extracted by pipetting 5 ml of
MACS buffer onto the column and firmly pushing the plunger into the column, flushing
out the labeled CD19-positive cells. B cells from all 6 buffy coats were combined. The
suspension was then spun down at 300 g for 5 minutes and resuspended in 10 ml of
16
CD40B medium. Cell concentration and total cell number were determined. An aliquot
of 1 x10 6 cells was assured for flow cytometric assessment of B cell purity and baseline
(0 hr) immune phenotyping.
2.1.5 NIH 3T3 HUCD40L CELL CULTURE
The adherent, mouse-derived NIH 3T3 fibroblast cell line transduced with human CD40
ligand (NIH huCD40L cells, provided by G. Freeman, DFCI, Boston, USA) was kept in
10 ml NIH medium in a 75 cm 2 cell culture flask. Stable expression of CD40L (CD154)
was monitored regularly by flow cytometry. Transduction also introduced a resistance
cassette conferring resistance to G418 (Genemicin), allowing selection for stably
transduced cells.
The cells were subcultured every 3-4 days. Medium was aspirated and the cells were
washed once in the flask with 10 ml of PBS. 5 ml trypsin/EDTA was then added and the
flask was incubated for 5 minutes at 37 °C to detach the adherent NIH cells. After
verifying that most cells were detached by light microscopy, 5 ml of NIH medium was
added to the flask to inactivate trypsin. The cell suspension was aspirated, transferred to
a 50 ml Falcon tube and centrifuged at 300 g for 5 minutes. After discarding the
supernatant, cells were washed again with 10 ml NIH medium and counted. For the
subculture, 2.5 x10 6 cells were transferred to a fresh flask and medium was added to a
total of 10 ml. To select for CD40L-expressing cells, G418 (Biochrom) was added to a
final concentration of 200 µg/ml.
The remaining cells were irradiated with 72 Gy at the blood bank of the University
Hospital of Cologne for use as feeder cells providing CD40L stimulation. After
irradiation, concentration of the cell suspension was adjusted to 0.1 x10 6 /ml with NIH
medium. The cells were placed on a six-well plate (Nunc), adding 2 ml (2 x10 5
cells)
per well. After 16 hours cells became adherent and could be used for coculture with
PBMCs or CD19-positive B cells.
2.1.6 HUMAN CD40-ACTIVATED B CELL CULTURES
Human CD40-activated B cells were generated following an established protocol. 100
Activation was induced by coculturing cells on a six-well plate with irradiated NIH
huCD40L fibroblasts prepared on the previous day (see 2.1.5).
17
The setup of gene expression and miRNA expression experiments is shown in Figure 1.
CD19-positive cells from 6 buffy coats were pooled yielding at least 160 x10 6 B cells.
An aliquot with 12 x10 6 cells was transferred to a separate tube for baseline (0 h)
analyses. From the remaining cells, two aliquots containing 60 x10 6 cells each were
made, representing the cells for the normoxic and hypoxic arms of the experiment. The
cells were spun down at 300 g for 5 minutes and resuspended in 60 ml of CD40B
medium to a concentration of 1 x10 6 cells/ml. Cells to be cultured in hypoxia were
resuspended in medium that had been pre-incubated in hypoxia over 24 hours to
equilibrate the oxygen content. IL-4 was added to the medium to a final concentration
of 50 I.E./ml.
For each time point (1 day, 4 days) and each experimental condition (normoxia and
hypoxia), activation of B cells was performed by coculturing 12 x10 6 B cells with NIH
huCD40L cells in 3 of 6 wells on one six-well plate. The remaining 3 wells contained
only NIH huCD40L cells which were used as controls for gene and microRNA
expression data. Thus, in total, 4 six-well plates with adherent NIH huCD40L cells were
required. In addition, wildtype NIH 3T3 cells were prepared to study B cells that had
not been activated. As B cells without stimulation go into apoptosis after 4 days and no
meaningful data can be generated at this time point, only 2 six-well plates were
prepared for the 1 day time point per normoxic and hypoxic condition.
Three wells on all six-well plates were washed once by aspirating the NIH medium and
then adding 2 ml of cold PBS to each well. After removing the PBS, plates for the
normoxic and hypoxic experimental arm were prepared. 4 ml of the B cell suspension (1
x10 6
cells/ml) were transferred to three wells on each plate, amounting to 12 x10 6 cells
per time point. 2 six-well plates with huCD40L and 1 six-well plate with wildtype NIH
3T3 were prepared for each of the normoxia and hypoxia experimental arms. Cell
culture plates to be incubated in hypoxia were placed into the hypoxia chamber
immediately. After adjusting the oxygen content in the chamber to 0.8% (see 2.1.1), all
six-well plates were transferred into the incubator.
1 day and 4 days after the start of the culture, B cells were harvested and prepared for:
(1) flow cytometry immunophenotyping (FACS), (2) cell lysis for subsequent RNA
extraction and microarray analysis. Additionally, culture supernatants were conserved
for future analyses of soluble factors.
18
Figure 1. Experimental setup for microarray analysis. Buffy coats from 3-4 individual donors were collected from the local blood bank. CD19+ B cells were isolated using
MACS (magnetic-activated cell sorting) positive selection microbeads. After enrichment, day 0 sample aliquot was preserved and the remaining cells were used for stimulation
cocultures: two cultures each for normoxia and hypoxia using NIH huCD40L, and one culture each for coincubation with NIH wildtype cells. 12 x10 6 cells were used per
culture. Cells were cultured for 1 day (CD40L and wildtype cocultures) and 4 days (CD40L coculture only) before further analysis.
w t-
Buffy coat
2.2.1 FACS ANTIBODIES
Flow cytometric analyses were comprised of 3 panels and 1 unstained sample (= 4 tubes
per condition). The following panels were used:
Table 2 FACS antibodies
FL2 PE CD24 BioLegend, Cat. 311106
FL3 ECD CD10 Beckman Coulter, Cat. IM3608U
FL4 PE Cy5.5 CD38 Beckman Coulter, Cat. A07780
FL5 PE Cy7 CD27 Beckman Coulter, Cat. A54823
FL6 APC CD21 BD Pharmingen, Cat. 555335
FL7 AlexaFluor700 CD19 Beckton Dickinson, Cat. 557921 Clone HIB19
FL8 APC H7 CD3 Beckton Dickinson, Cat. 641397
FL9 Pacific Blue CD20 BioLegend, Cat. 302328
FL10
FL3
FL5 PE Cy7 CD27 Beckman Coulter, Cat. A54823
FL6 APC CD21 BD Pharmingen, Cat. 555335
FL7
FL9 Pacific Blue CD20 BioLegend, Cat. 302328
FL10
FL3 ECD CD62L Beckman Coulter, Cat. IM2713U
FL4 PC 5.5 CD38 Beckman Coulter, Cat. A07780
FL5 PC7 CD27 Beckman Coulter, Cat. A54823
FL6 APC CCR10 R & D, Cat. FAB3478A
FL7
FL9 Pacific Blue CD20 BioLegend, Cat. 302328 Clone 2H7
FL10
20
2.2.2 FACS CELL PREPARATION
For immune phenotyping, cell samples were analysed at 0 hours, 4 hours, 1 day, and 4
days. B cells from 3 wells (= 12 ml cell suspension) of each of the four culture
conditions (normoxic CD40-activated, normoxic non-activated, hypoxic CD40-
activated, hypoxic non-activated) were transferred into a 50 ml tube. From each tube,
four 1 ml aliquots of cell suspension were transferred into four 5 ml round bottom
FACS tubes (Sarstedt, Cat. 55.1579) for FACS analyses. If the remaining cells were
scheduled for gene or microRNA expression analyses, the remaining suspension was
spun down (226 g, 5 min.) for RNA extraction (see 2.3.1). The supernatant was
transferred into 1.8 ml cryotubes (Nunc) and stored at -80 °C for future analyses.
Aliquots of cells for FACS (1 x 10 6 cells per panel) were spun down in FACS tubes and
washed twice with PBS at 353 g for 5 minutes. 1.5 µl of each antibody were added to
the cell pellet. After an incubation period of 15 minutes at 4 °C in the dark, cells were
washed with cell wash solution (Beckton Dickinson, Cat. 349524) and resuspended in
300 µl of the same solution. FACS analyses were performed on a 10-color Gallios flow
cytometer (Beckman Coulter) using the appropriate compensations. Subsequent analysis
of the FACS data was performed with Kaluza ver. 1.3 (Beckman Coulter).
2.3 MICROARRAY ANALYSES
2.3.1 CELL LYSIS, RNA EXTRACTION AND QUALITY CONTROL
After removing cell aliquots for FACS analyses (see above), the remaining cells from
each time point and culture condition (approx. 8 x10 6 cells each) were spun down at 226
g for 5 minutes. Cell pellets were kept on ice to protract potential changes in gene
expression in response to reoxygenation. The cells were lysed in the same 50 ml tube as
soon as possible using 2 ml of Trizol reagent (Invitrogen, #15596018). The cell lysate
was aliquoted into two portions of 1 ml and stored in 1.5 ml Eppendorf tubes at -80 °C
(Sanyo) until further processing. The NIH feeder cells were lysed in 1 ml of Trizol only
and the lysate was stored in one 1.5 ml tube.
RNA from all samples was extracted using the chloroform-phenol extraction method
established by Chomczynski et al. 25
. All procedures requiring RNA handling were
performed with sterile pipette tips (SafeSeal Tips Professional, Biozym, Cat. 770005
(10 µl), 770200 (200 µl), 770400 (1000 µl)) and sterile Eppendorf tubes. Trizol lysates
21
were thawed at room temperature. 200 µl of Chloroform (Sigma Aldrich) were added to
each sample and the tube was inverted for approx. 45 sec. until the suspension turned
opaque and homogenous. The tubes were then centrifuged for 10 minutes at 13000 rpm
in a benchtop microcentrifuge (Eppendorf fresco) which had been pre-chilled to 4 °C.
The clear supernatant (approx. 450 µl) containing RNA was transferred into a fresh 1.5
ml tube (Eppendorf). 500 µl of Isopropanol was added to the tube to facilitate
precipitation. Following 10 minutes incubation at room temperature, tubes were
centrifuged at 13000 rpm for 30 minutes. After centrifugation RNA became visible as a
small white pellet at the bottom of the tube. Supernatant was discarded and the RNA
pellet was washed with 500 µl of Ethanol 80% (Sigma Aldrich). Tubes were centrifuged
at 13000 rpm for 30 minutes and supernatant was discarded. The washing step was
repeated once. After the second washing, the pellets were exposed to the air by
removing all liquid remains with a pipette. Tubes were flipped upside down and placed
on tissue paper, allowing the pellet to dry. RNA was dissolved in 33 µl of RNAse-free
ddH2O by heating samples to 55 °C for 5 minutes. RNA concentration, 230/260 and
260/280 ratios were measured using a NanoDrop N1000 photometer (PeqLab). For
microarray experiments, only RNA samples with a 260/280 ratio between 1.6-2.1 and a
260/230 ratio above 1.5 were used.
To verify RNA integrity, agarose gel electrophoresis was performed. 1.5 % agarose gel
was prepared by adding 750 mg of agarose (Sigma Aldrich) to 50 ml of TAE buffer
(Gibco). The buffer was heated in a microwave until agarose was completely dissolved.
5 µl of ethidium bromide was added and the agarose solution was poured into a gel
casting mold. The solution was allowed to cool down and solidify.
250 ng of RNA was aliquoted and RNAse-free ddH2O was added to a volume of 5 µl. 5
µl of 2x loading dye (Thermo Scientific) was added to a final volume of 10 µl. Samples
were heated to 95 °C for 5 minutes before loading onto the gel. The samples were run at
120 V for 30 minutes against an RNA ladder (Thermo Scientific, Cat. SM1823).
Visualization of bands was achieved using a UV-transilluminator (Bio-Rad) equipped
with a digital camera and appropriate capture software. Intact RNA shows two distinct
bands at 5000 and 2000 bases, corresponding to the 28S and 18S rRNA. Completely
degraded RNA appears as a low molecular weight smear. Contamination of RNA with
genomic DNA (gDNA) shows as an additional, high molecular weight band above the
28S rRNA band (see Figure 2).
22
A B C
Figure 2. (A) Intact bands in lanes 1-3. (B) degraded RNA band in lane 3. (C) gDNA-contaminated
RNA bands in lanes 1-3. M: RNA size marker
2.3.2 GENE EXPRESSION PROFILING
Total RNA was generated from 5 independent CD40B cell cultures derived from 6
donors each (see 2.1.6). Samples from 4 hours, 1 day and 4 days were taken and RNA
was isolated following the protocol outlined in 2.3.1. To measure gene expression levels
in activated B cells cultured in normoxia and hypoxia, 2 gene expression microarrays by
Illumina (HumanHT-12 v4 Expression BeadChip) were used. For gene expression
profiling, RNA from cells exposed to the following conditions was used:
Table 3 Gene expression profiling samples
Culture time Activation stimulus Oxygen Arrays used
0 hour native normoxia 3
1 day CD40-activated normoxia 3
1 day CD40-activated hypoxia 3
1 day non-activated normoxia 3
1 day non-activated hypoxia 3
4 day CD40-activated normoxia 3
4 day CD40-activated hypoxia 3
Per condition, RNA from the 5 experiments was combined to yield 3 samples that were
eventually loaded onto the BeadChips as biological triplicates. Triplicate sample 1
(named “P23”) consisted of equal amounts of RNA from experiments 1 and 2, triplicate
sample 2 (named “P46”) contained RNA from experiments 3 and 4. Triplicate sample 3
(named “P7”) consisted of RNA from experiment 5 only. Each sample contained 1000
ng of total RNA, and the volume was adjusted to 100 µl with ddH2O.
1 2 3 M M
1 2 3 1 2 3 4
23
RNA CLEANUP
To desalt and purify the RNA, samples were cleaned up using the RNeasy MinElute
Cleanup kit (Qiagen, #74204). Total RNA is bound to a silica membrane and
contaminants as well as smaller RNA fragments are washed away. RNA can then be
eluted in small volumes. With this procedure, RNA fragments longer than 200
nucleotides were purified excluding smaller rRNAs and tRNAs. 350 µl of the supplied
RLT buffer and 250 µl of 100% ethanol were added to the 100 µl RNA samples. The
solutions were added to a MinElute spin column containing a silica membrane. The
columns were placed in an Eppendorf tube and centrifuged for 15 seconds at 8000 g in a
benchtop microcentrifuge (Eppendorf fresco). Flow-through was discarded and the
columns were washed once by adding 500 µl RLT buffer followed by centrifugation for
15 seconds at 8000 g. Flow-through was discarded and 500 µl of 80% ethanol were
transferred onto the columns. Columns were centrifuged for 2 minutes at 8000 g.
Eluates were discarded and the columns were dried by centrifugation for 5 minutes at
8000 g. Columns were placed in fresh Eppendorf tubes and 14 µl of ddH2O were
transferred directly onto the membrane. Eluation of RNA was facilitated by
centrifugation for 1 minute at 8000 g. RNA concentrations were subsequently
determined on a NanoDrop 1000 spectrophotometer (PeqLab).
CDNA SYNTHESIS AND BIOTIN-LABELED CRNA SYNTHESIS
cDNA synthesis and in vitro transcription was performed with the TargetAmp Nano
Labeling Kit for Illumina Expression BeadChip (Epicentre, #TAN07924). Aliquots of
samples containing 100 ng of purified RNA in 2 µl were prepared in 100 µl reaction
tubes (Eppendorf). Master mixes for 1 st and 2
nd strand synthesis were made for 22
samples (7 conditions in triplicate plus 1 spare, see Table 4).
Table 4 cDNA synthesis master mixes
1 st strand master mix
Reagent per 1 sample for 22 samples
1 st strand cDNA premix (supplied with kit) 1.5 µl 33 µl
Dithiothreitol (DTT) 0.25 µl 55 µl
SuperScript III Reverse Transcriptase
2 nd
strand cDNA premix (supplied with kit) 4.5 µl 99 µl
2 nd
strand DNA polymerase (supplied with kit) 0.5 µl 11 µl
∑ 5.0 µl 110 µl
1 µl of T7-oligo(dT) primers (supplied with kit) were added to each sample and primers
were allowed to anneal at 65 °C in a thermocycler (Biometra) for 5 minutes. Samples
were then cooled on ice for 1 minute. After addition of 2 µl of 1 st strand master mix,
strand synthesis was performed at 50 °C for 30 minutes. Immediately after 1 st strand
cDNA synthesis, 5 µl of 2 nd
strand master mix were added to the samples. Tubes were
gently mixed and were incubated at 65 °C for 10 minutes. To stop the reaction, the
tubes were incubated at 80 °C for 3 minutes and stored at 4 °C until further processing.
In the next step, biotin-labeled cRNA was generated in an in vitro transcription reaction
using biotin-labeled UTP. The reaction master mix was prepared as follows:
Table 5 IVT reaction master mix
Reagent per 1 sample for 22 samples
T7 transcription buffer (supplied with kit) 2.0 µl 44 µl
UTP / biotin-UTP (supplied with kit) 3.0 µl 66 µl
NTP premix (supplied with kit) 10 µl 220 µl
T7 RNA polymerase (supplied with kit) 2.0 µl 44 µl
Dithiothreitol (DTT) (supplied with kit) 3.0 µl 66 µl
∑ 20 µl 440 µl
20 µl of IVT reaction master mix was added to each tube. Reaction was carried out in a
thermocycler at 42 °C for 4 hours. After transcription, remaining cDNA was digested
with 2 µl DNAse I (supplied with kit) at 37 °C for 15 minutes. cRNA was isolated from
the reaction mix using the RNeasy MinElute Cleanup kit (Qiagen, #74204; see 0).
Integrity of cRNA was verified by agarose gel electrophoresis. Intact cRNA shows as a
broad, undefined band up to 2000 bases in size, representing a wide range of different
transcripts.
M 1 2 3
HYBRIDIZATION TO BEADCHIP AND STAINING
A defined amount of cRNA was prepared for each sample and loaded onto the
BeadChips for hybridization. Following washing, chips were incubated with a
streptavidin-Cy3 conjugate leading to fluorescent labeling of biotin-containing cRNA
samples.
750 ng of cRNA from each sample was was aliquoted and RNAse-free ddH2O was
added to a volume of 5 µl. 10 µl of HYB buffer (Illumina) was added to the sample and
the tube was vortexed and spun down briefly. BeadChips were placed in an Illumina
hybridization chamber filled with 200 µl HCB buffer (supplied with BeadChip) for
humidification. 15 µl of biotin-labeled cRNA was loaded onto each BeadChip inlet port.
After loading all samples, hybridization chambers were sealed and incubated in an
Ilumina hybridization oven at 58 °C overnight (16 hours). The following day,
BeadChips were removed from the chamber and submerged in a beaker containing 1
liter of E1BC wash buffer (Illumina). Coverseals were removed from the chips while
keeping them submerged in the buffer. BeadChips were transferred to a slide rack
(Illumina) submerged in a staining dish containing 250 ml E1BC washing buffer and
washed gently. The rack was then placed in a waterbath containing 500 ml of high
temperature wash buffer heated to 55 °C and incubated for 10 minutes. After incubation,
chips were washed with E1BC buffer again at room temperature for 5 minutes. The rack
was then transferred to a fresh staining dish containing 250 ml of 100% ethanol. After
10 minutes, chips were briefly washed in E1BC buffer and then placed into a wash tray
(Illumina) containing 4 ml of E1 blocking buffer (Illumina). Blocking was performed
for 10 minutes. After blocking, chips were transferred to a fresh wash tray containing 2
ml of block E1 buffer with a 1:1000 dilution of Cy3-streptavidin (stock of 1 mg/ml,
Illumina). After a 10 minute incubation time, chips were washed in a fresh staining dish
containing 250 ml E1BC washing buffer for 5 minutes. The rack containing the chips
was then centrifuged in an appropriate holder (Illumina) at 275 g for 4 minutes allowing
the slides to dry. After centrifugation, chips were stored in the dark until imaging.
26
BEADCHIP DATA AQUISITION
Data aquisition was performed on an Illumina BeadArray reader using the supplied
BeadScan software (Ilumina). Decode maps were obtained for each chip with the
provided download utility (Ilumina) and loaded to the BeadScan software prior to
scanning. Scanning was performed directly after placing BeadChips into the reader.
Raw array data was processed and quantil-normalized using the gene expression module
of the GenomeStudio software suite (Illumina).
2.3.3 MICRORNA EXPRESSION PROFILING
For miRNA expression analyses, 5 Illumina Sentrix BeadChip arrays (Universal-12
BeadChip, 1536 BeadTypes, Illumina, Cat. 11288011) were used. The assay panel
contains 1,145 different probes, representing the majority (approx. 61%) of known
miRNAs. 49, 87
Each chip holds 12 slots for sample analysis.
First, 280 ng of total RNA were polyadenylated with poly-A polymerase.
Polyadenylated RNA was then converted to cDNA using a biotinylated deoxythymidine
(dT) primer containing an universal PCR primer sequence at its 5’-end. Biotinylated
cDNA was hybridized to miRNA-specific oligos (MSO) containing a microRNA-
specific sequence, a universal PCR primer sequence, and an address sequence for
hybridization to the array. The assay contained MSOs for each miRNA interrogated on
the array. After allowing cDNA and the corresponding MSO to hybridize, the hybrid
was immobilized using streptavidin beads linked to paramagnetic particles.
Nonhybridized cDNA was washed away. Amplification was then carried out by
polymerase chain reaction (PCR) using a fluorescently labeled primer. The PCR
product was then hybridized to the array.
Sample preparation of total RNA for miRNA expression profiling differed from gene
expression array sample preparation outlined in 2.3.2. The key differences between both
procedures are that for miRNA expression profiling, the cDNA derived from miRNAs
is specifically targeted and immobilized for amplification and the remaining cDNA is
removed from the reaction. For gene expression profiling, total cDNA was amplified in
the in-vitro transcription step. Also, labeling of cDNA was performed using a
fluorophore-linked primer instead of a streptavidin-Cy3 conjugate.
27
RNA from cells in the following conditions was used for miRNA expression profiling:
Table 6 miRNA expression profiling samples
Culture time Activation stimulus Oxygen Arrays used
0 hours native normoxia 4
1 day CD40-activated normoxia 5
1 day CD40-activated hypoxia 5
1 day non-activated normoxia 5
1 day non-activated hypoxia 5
4 day CD40-activated normoxia 5
4 day CD40-activated hypoxia 5
1 day and 4 day samples were not pooled but were individually loaded onto one single
array, amounting to 30 arrays. Thus, there were 5 biological replicates available for each
of these conditions. For the 0 hour samples, RNA from experiment 1 and 2 were
combined and RNA from each experiment 3-5 were individually loaded onto one array,
totaling 4 arrays. Volume was adjusted to 7 µl per sample using ddH2O.
POLYADENYLATION OF TOTAL RNA AND CDNA SYNTHESIS
The sample containing 280 ng total RNA was added to 5 µl of PAS reagent (supplied by
Illumina) on a 96 well plate. After sealing, the plate was centrifuged at 250 g for 1
minute. Subsequently, the plate was incubated at 37 °C for 60 minutes to facilitate
polyadenylation. The plate was then transferred to a heat block and incubated at 70 °C
for 10 minutes to inactivate the enzyme. After incubation, the plate was centrifuged at
150 g for 1 minute. For subsequent cDNA synthesis, 8 µl of CSS reagent (Illumina) was
added per sample. The plate was centrifuged briefly at 250 g and samples were
incubated at 42 °C for 60 minutes. To stop the reverse transcriptase reaction, the plate
was then transferred to a heat block and incubated at 70 °C for 10 minutes.
CDNA HYBRIDIZATION TO MIRNA-SPECIFIC OLIGOS (MSO)
In this step, miRNA-specific oligos (MSOs) for each miRNA sequence target of interest
were annealed to the biotinylated cDNA. The hybrid is then captured by paramagnetic
particles.
5 µl of MAP and 30 µl of OB1 (Illumina) was added to 15 µl of biotinylated cDNA and
the total volume was transferred to a fresh 96-well microwell plate. The plate was
centrifuged at 250 g for 1 minute. Samples were then placed into a heat block set to
70 °C. Immediately after placement, the heat block was set to 40 °C and allowed to cool
28
down over 4 hours, facilitating hybridization of cDNA and MSOs. After annealing, the
microwell plate was placed on a magnetic plate (Ilumina) until the paramagnetic beads
were completely captured at the bottom of the well, forming a small visible pellet. The
supernatant was discarded carefully. 50 µl of AM1 buffer (Illumina) was added to the
wells and the beads were resuspended by vortexing at 1600 rpm for 20 seconds.
Washing of the beads was repeated once. AM1 buffer washing was followed by
washing with UB1 (part of Illumina supplied reagents) for two times. Finally, the beads
were resuspended in 37 µl of MEL reagent (Ilumina). This reagent contains a
polymerase and ligase for extension of the cDNA-MSO template. The microwell plate
was then incubated at 45 °C for 15 minutes.
AMPLIFICATION OF MIRNA-DERIVED CDNA
During the extension and ligation step, a fresh 96-well plate containing reagents for
PCR amplification was prepared. 64 µl of DNA polymerase (Illumina, included in
assay) and 50 µl of uracil DNA glycosylase (Illumina) were added to a tube containing
800 µl of SCM reagent (Single Color Master Mix, Illumina). 30 µl of the mixture was
then applied to each well of the PCR plate.
The plate containing the cDNA-MSO template was removed from the heat block and
placed on a magnetic plate. The beads at the bottom of the wells were captured and the
supernatant was discarded carefully. Beads were washed once with 50 µl of UB1
reagent and resuspended in 35 µl of IP1 (Inoc PCR 1 Reagent, Illumina). After
incubation at 95 °C for 1 minute, beads were captured on the magnetic plate and 30 µl
of supernatant from each well was transferred to a fresh well containing PCR reagents
on the PCR plate. After sealing the plate, the PCR reaction was carried out according to
the following conditions:
Temperature Time
56 °C 35 seconds
72 °C 2 minutes
72 °C 10 minutes
4 °C 5 minutes
During the cycles, the templates were amplified to generate fluorescently labeled PCR
products. The SCM master mix contained two universal primers for template
amplification: one of the primers was biotinylated and the other was labeled with a
fluorescent dye. This allows both strands to be separated during the following steps.
After the amplification reaction, 20 µl of MPB reagent (magnetic particle B, Illumina)
were added to the PCR products, binding the double-stranded PCR products to
paramagnetic beads via the biotinylated strand. The samples were then transferred to
corresponding wells on a filter plate (Illumina). The plate was sealed and incubated at
room temperature for 1 hour under light protection, allowing the PCR products to bind
to the filter membrane. After incubation, the filter plate was placed on a waste plate and
centrifuged at 1000 g and 25 °C for 5 minutes. 50 µl of UB2 (universal buffer 2,
Illumina) was added to the filter membranes. The plate was again centrifuged at 1000 g
and 25 °C for 5 minutes. After adding 30 µl of MH1 reagent (make Hyb 1 reagent,
Illumina) and 30 µl of 0.1 M NaOH (Sigma Aldrich) to the wells, the filter plate was
placed onto a fresh plate for elution of the single stranded, fluorescently labeled PCR
product. Elution was performed by centrifuging the plate at 1000 g and 25 °C for 5
minutes.
HYBRIDIZATION TO BEADCHIP
Each array on the BeadChips was loaded with 15 µl of sample. The hybridization
process was performed in hybridization chambers supplied by Illumina. 200 µl of CHB
(chamber humidification buffer, Illumina) was added to the chamber to provide
humidity during the hybridization process. Each chamber can accommodate 4
BeadChips. Hybridization was performed at 60 °C in an Illumina Hybridization Oven
for 30 minutes. After that, the temperature was adjusted to 45 °C and hybridization was
continued for 16 hours overnight. Following incubation, cover seals were removed and
the chips were washed three times for 5 minutes with UB2 (universal buffer 2, Illumina)
and XC4 (XStain BeadChip 4, Illumina) reagents in a 50 ml centrifuge tube. Chips were
entirely submerged in buffer during each washing step. Subsequently, the arrays were
dried in vacuum desiccators at 0.68 bars for 50 minutes (supplied by Illumina).
BEADCHIP DATA AQUISITION
30
Data aquisition was performed on an Illumina BeadArray reader using the supplied
BeadScan software (Ilumina) analogous to the steps outlined in 0.
2.3.4 MICROARRAY DATA ANALYSIS
The first aim of the array analysis was to identify genes in CD40-activated or non-
activated B cells that were significantly regulated under hypoxic conditions. In addition,
these regulated genes should have a functional relation to the immunologic capacity of
the B cell. Finally, differentially expressed miRNAs were computationally matched
against the regulated genes to identify potential interactions.
COMPARATIVE ANALYSIS WITH DCHIP
Quantile-normalized data was loaded into dChip software (DNA-Chip Analyzer 64 bit,
build 04/13/2010; Cheng Li and Wing Wong labs) on a personal computer (Dell
Precision workstation T3400) for comparative analyses. Biological replicates from each
condition were combined and defined as sample groups. Thresholds for differential
expression were defined as follows:
Table 8 Gene and miRNA differential expression cutoffs
Fold change p
Result lists were saved as Microsoft Excel spreadsheet files.
FUNCTIONAL CLUSTERING OF REGULATED GENES
Genes that were found to be differentially expressed in hypoxia were matched to known
and experimentally validated gene sets using the Molecular Signatures Database
(MSigDB, Broad Institute, http://www.broadinstitute.org/gsea/msigdb/annotate.jsp).
Immunologically relevant gene sets in the database were scanned for genes that were
differentially expressed in the experiment.
The lists of differentially expressed genes were copied into the form and the ‘canonical
pathways’ checkboxes “CP: BIOCARTA”, “CP: KEGG” and “CP: REACTOME” were
checked to match the submitted gene lists against the gene sets. ‘Show genesets’ was set
to top 50.
Matching analysis with regulated miRNA and downregulated mRNA was performed
using the microRNA and mRNA Integrated Analysis platform (MMIA). 115
A list of the
miRNA regulated in CD40B cells or non-activated B cells was copied into the MMIA
form in step 1. In step 2, the target algorithm was set to TargetScan 5.1 (Whitehead
Institute for Biomedical Research, Boston, USA). 97
The corresponding gene expression
file containing a list of all regulated genes was uploaded in the next step. The
expression values in the control and experiment groups were set to 1 and 0.5,
respectively (as suggested in MMIA’s documentation). Preprocessing was disabled and
fold-change set to 1.5. The remaining settings were left at default.
As a result, a list of predicted targets was returned. These genes were downregulated in
hypoxic CD40-activated B cells and showed potential binding sites for the miRNAs that
were upregulated in the sample. To functionally cluster the miRNA target genes, a gene
set enrichment analysis was performed using the same settings described in 0. Genes
that were represented in an immunologically relevant gene set were selected.
2.3.5 STATISTICS
Arithmetic mean und standard deviation values were calculated in Microsoft Excel 2007
spreadsheets. GraphPad Prism software version 5 was used to determine significance
levels using the statistical tests indicated. P-values less than 0.05 were deemed
statistically significant. Figures were also created in GraphPad Prism.
32
3.1 IMMUNE PHENOTYPING
B cell purity was measured before initiating each CD40B cell culture. After CD19 +
MACS positive selection, average B cell (CD19 + CD20
+ ) enrichment was 95,9% (SD
±2,3%) of all lymphocytes. Binding of fluorescent CD19 + antibody, however, might
have been impaired by the MACS anti-CD19 + antibody from the enrichment procedure,
leading to a weaker signal during FACS. When gating for CD20-positive cells, purity
was 96,9% (SD ±1,1%) of lymphocytes after enrichment.
3.1.1 HYPOXIC CD40BS ARE REDUCED IN CELL SIZE
Purified B cells were cultured for 1 day and 4 days and relative cell volume of CD40-
activated B cells was measured by flow cytometry in 4 experiments. Median forward
scatter (FSC) values of CD19 + CD20
+ B cells were analyzed (Figure 4A and B).
After 1 day of CD40 activation, cell volumes of activated B cells did not change
significantly compared to native B cell baseline volumes (0 hours). After 4 days of
CD40 activation, cell size of CD40B cells cultured in hypoxia were significantly
smaller than in cells cultured in normoxic conditions (p=0.03). Median FSC increased
2.1-fold compared to baseline values in normoxic B cells, while hypoxic B cells showed
an increase by only 1.3-fold (Figure 4C).
Cell morphology was evaluated by conventional light microscopy and showed
differences in cell growth between oxygen conditions as well. Cells cultured in
normoxia for 4 days formed large, round cell clusters as a result of homotypic adhesion,
initiated by CD40 activation and upregulation of adhesion molecules (Figure 4D, lower
left image). Cells cultured under hypoxic conditions formed, if at all, markedly smaller
cell clusters, featuring less round and more random-shaped and elongated phenotypes
after 4 days (Figure 4D, lower right image). To measure cell proliferation, cell
concentrations in CD40B cultures were evaluated after 4 days. As suggested by the
above results, concentrations in hypoxic CD40B cultures were significantly lower than
in normoxia. Over the course of 4 days, normoxic CD40B cells proliferated by 1.6-fold
(1 x10 6 to 1.6 x10
6 /ml), whereas hypoxic CD40B cell concentration remained stable (1
x10 6 to 0.9 x10
6 /ml; p≤0.01 using Student’s paired t-test).
33
Figure 4. A. Evaluation of relative cell volumes by flow cytometry. Viable cells staining positive for
CD19+ and CD20+ B cells were gated and a scatter plot with forward vs. sideward scatter was created at
0 and 4 days. Representative experiment shown. B. Representative experiment of CD19+CD20+ cells
after 4 days culture. FSC was displayed in a histogram. C. Dot plot of median FSC values from CD40B
cultures (n=4). D. Images from normoxic and hypoxic cell cultures using phase contrast microscopy at
the time points shown. 100x magnification. E. Dot plot of CD40B cell concentrations before and after 4
days of culture in normoxia and hypoxia. Black bar represents arithmetic mean. Error bars represent
standard deviations (SD). * p≤0.05; ** p≤0.01; paired Student’s t-test.
34
3.1.2 HYPOXIC CD40BS EXPRESS LESS CD80 AND CD86
CD80, CD86 and HLA-DR are stimulatory molecules required for T cell activation by
antigen-presenting cells. 44, 102
Expression of these molecules was measured after
culturing CD40Bs in normoxic and hypoxic conditions for 1 and 4 days, followed by
flow cytometric analysis (see 2.2, Figure 5A). This experiment was repeated 5 times
with individual donors. Expression of CD80 and CD86 was significantly repressed in
CD40Bs cultured in hypoxic conditions for 1 or 4 days, as measured by median
fluorescence intensity (MFI CD80: 12.6 vs. 8.1, CD86: 262.0 vs. 124.0 on day 4 in
norm- and hypoxia, p≤0.01, Figure 5C). Similarly, the CD80 + CD86
+ activated B cell
population was decreased on day 1 (21.4% vs. 17.5% of CD19 + CD20
+ , p≤0.01) and day
+ , p=0.01, Figure 5E). Expression of HLA-DR was
higher in CD40Bs after 4 days of culture in hypoxia, but differences failed to reach
significance (p=0.12, Figure 5C).
3.1.3 ALTERED EXPRESSION OF ADHESION MOLECULES CD62L AND CD54
Expression of adhesion molecules on the cell surface is a feature of B cell activation and
is required for homing of these cells to tissues such as secondary lymphoid organs, i.e.
lymph nodes. 134
L-selectin (SELL, CD62L) was evaluated in normoxic and hypoxic conditions (Figure
5B). Expression of CD54 was induced in both normoxic and hypoxic B cells upon
CD40 activation. However, median fluorescence intensities in the CD54 channel were
slightly increased in hypoxic B cells on day 4 (39 vs. 43, p≤0.01, Figure 5D),
suggesting upregulation of this protein under hypoxia. Conversely, CD62L was
downregulated in hypoxic CD40B cells on day 4 (22 vs. 11, p=0.03, Figure 5D).
Interestingly, when compared to baseline expression on day 0, expression of CD62L
was markedly downregulated in normoxia as well as hypoxia after one day of CD40
activation, a finding not observed in non-activated B cells.
35
36
Figure 5. Expression of surface molecules. A. Live cells were identified in FSC/SSC scatter plot and
selected for CD19+ positivity. Expression of CD80, CD86 and HLA-DR immunostimulatory molecules
were displayed in histograms at the indicated time points. Representative experiment shown.
Blue lines -: normoxic cells, red lines -: hypoxia, grey lines -: unstained controls. B. Histograms of CD54,
CD62L adhesion molecule expression on CD19 + cells at indicated time points. Representative experiment
shown. C. Median MFI of indicated immunostimulatory molecules from CD40B (+CD40L) and non-
stimulated (-CD40L) B cell cultures (n=5). D. Median MFI of indicated adhesion molecules B cell
cultures (n=4). E. Dot plot showing percentage of CD80 + CD86
+ activated B cells in CD19+CD20+ B
cells. Blue circles : normoxic cells, red squares : hypoxic cells. Black bar represents arithmetic mean.
Error bars represent standard deviations (SD). * p≤0.05; ** p≤0.01; paired Student’s t-test.
3.1.4 SHIFTS IN B CELL SUBPOPULATIONS UNDER HYPOXIA
Analysis of subsets was performed using B cell differentiation markers IgD and CD27
(Figure 6A). After 4 days of culture in hypoxia, the mature naïve IgD + CD27
- population
of CD40 B cells was significantly smaller compared to the normoxic counterpart (33%
-
- cell compartments were larger under hypoxic conditions,
suggesting a shift from naïve to memory B cell phenotype under hypoxic CD40
stimulation. Analysis of the plasma cell and plasmablast population showed an overall
decrease of IgD - CD27
hi cells upon CD40 activation in both normoxic and
hypoxic cultures. The decline in the frequency of these cells was more pronounced
under hypoxic conditions and reached significance after 24 hours (1.1% vs. 0.2% in
norm- and hypoxia, p=0.04, Figure 6C). The size of the CD24 hi
CD38 hi
regulatory B cell
population increased in normoxic and hypoxic CD40B cell cultures on day 4 compared
to day 1. However, the expansion of this B cell subset after 4 days was less distinct in
hypoxia than in normoxia, resulting in a significant difference between both conditions
on day 4 (2.4% vs. 1.7% in norm- and hypoxia, p=0.03, Figure 6D).
37
Figure 6 A. Evaluation of B cell subsets by flow cytometry. Viable, CD19+ B cells were selected and
expression of maturation markers IgD and CD27 was evaluated in a scatter plot after 0 hours and 4 days.
Percentages of each IgD and CD27 subset are displayed. Representative experiment shown. B.
Percentages of naïve and memory B cell subsets of CD19+ CD40B cells (n=4). C. Percentages of the
CD27 hi
CD38 hi
- cells. D. Percentages of the
CD24 hi
CD38 hi
regulatory B cell populations of CD19 + cells. Blue circles : normoxic cells,
red squares : hypoxic cells. Black bar represents arithmetic mean. * p≤0.05; ** p≤0.01; paired
Student’s t-test
With hierarchical clustering analysis, groups of samples with similar expression patterns
can be defined. Hierarchical clustering was performed to check data quality and verify
that arrays representing biological replicates clustered together. Before clustering gene
expression arrays, expression values of all arrays were log2-transformed to avoid
skewness effects by outliers. Gene expression data was then subjected to filtering to
find signature genes that were differentially expressed in one of the samples: Only
genes showing a coefficient of variation (standard deviation/mean) between 1 and 1000
were selected to avoid noise by non-changed data sets. 597 genes were selected after
filtering with these criteria. These genes became the basis for hierarchical clustering of
samples (Figure 7).
Clustering of CD40B samples (CD40B cultures 1 day, 4 days; in normoxic and hypoxic
conditions) did match the experimental setup: the biological replicates (triplicates),
cultured for the same time in the same oxygen condition, showed closest similarity to
each other compared to other samples and clustered together on the first level (all
p≤0.01). In the superordinate hierarchy level, samples from the same culture time (1 day
and 4 days) were grouped together (p≤0.001). This indicates that culture time had a
more profound impact on quantitative transcriptional changes than oxygen conditions.
Triplicates from native B cells on day 0 also clustered together (p≤0.01). There was one
irregularity in the samples from non-activated B cells: only 2 of the day 1 non-activated
B cells triplicate samples (P23_NANO and P46_NAHY) for norm- and hypoxia did
cluster on the first level (Figure 7). The third replicate clustered with its hypoxic
counterpart, and this couple itself grouped with day 0 native B cell samples. This may
be explained by the composition of the outlying sample (P7), which contained material
from five individuals. The other replicates (P23 and P46) consisted of pooled material
from ten individuals, representing a more homogenous sample and alleviating potential
confounding outlier effects.
Figure 7. Hierarchical clustering of gene expression data. Red: upregul