Transcript FULLY FUNCTIONAL IMMUNE ORGAN GROWN IN MICE FROM

FULLY FUNCTIONAL IMMUNE
ORGAN GROWN IN MICE FROM
LAB-CREATED CELLS
Maleeha Akram
08-arid-1772
Ph.D. Scholar
Department of Zoology
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Contents

Thymus
◦ Structure and Function
◦ Types
◦ Dysfunctions and Treatment
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Thymic involution
Generation of TECs
Functional attributes of iTECs
iTECs can form functional thymus
Conclusion
Reference
Thymus
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The thymus is
◦ a soft
◦ roughly triangular in shape
◦ specialized
organ
of
the immune system

It is located
◦ in the mediastinum of the
thoracic cavity between
the lungs
◦ anterior and superior to
the heart
◦ posterior to sternum
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Structure of thymus
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The thymus is of a pinkish-gray
color and lobulated on its
surfaces.
It has two distinct but identical
lobes
– each surrounded by a tough, fibrous
capsule.
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Within each lobe is a superficial
region of tissue called the cortex
and a histologically distinct
deep region called the medulla.
Epithelial tissues and lymphatic
tissues containing dendritic cells
and macrophages make up the
majority of both regions of the
thymus.
Cortex
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The cortical portion is
mainly
composed
of thymocytes (developing
T-lymphocytes)
Supported by a network of
finely-branched epithelial
reticular cells, which is
continuous with a similar
network in the medullary
portion.
This
network
forms
an adventitia to the blood
vessels.
Thymocytes
Reticular
cells
Medulla
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The
medulla
is
continuous
between
adjacent lobules
The medulla is paler
staining, less densely
cellular than the cortex
and contains
– More mature T-cells
– Prominent epithelial cells
– Hassalls corpuscles
– Admixed macrophages
– Dendritic cells
– B lymphocytes
Thymus function

The thymus serves a vital role in the training and
development of T-lymphocytes or T cells.
◦ T cells defend the body from potentially deadly
pathogens such as bacteria, viruses and fungi.
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The function of the thymus is to receive
immature T cells
– that are produced by hematopoietic stem cells (HSC)
in the red bone marrow
– and train them into functional, mature T cells that
attack only foreign cells.
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It also secretes the hormone thymosin that
stimulates the development of T-cells
Immature T
Lymphocytes
Thymus
Mature T-cells
Thymosin
Stimulates
• Development of T-cells
• Other immune cells
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Types of T cells

Helper T cells (TH cells)
◦ Assist other white blood cells in immunologic processes,
 maturation of B cells into plasma cells and memory B cells,
 activation of cytotoxic T cells and macrophages.
◦ Also known as CD4+ T cells because they express
the CD4 glycoprotein on their surfaces.
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Cytotoxic T cells (TC cells)
◦ Destroy virus-infected cells and tumor cells, and are also
implicated in transplant rejection.
◦ These cells are also known as CD8+ T cells since they
express the CD8 glycoprotein at their surfaces.
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Process of T-cell maturation
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T cells first reside within the cortex of the
thymus
– where they come in contact with epithelial cells
presenting various antigens.
The immature T cells that respond to the
antigens corresponding to foreign cells are
selected to survive, mature and migrate to the
medulla
 The rest die via apoptosis and are cleaned up
by macrophages. This process is known as
positive selection.
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Continued…
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Upon reaching the medulla
– the surviving T cells continue to mature and are
presented with the body’s own antigens.
T cells that bind to the body’s own antigens test
positively for autoimmunity.
 Autoimmune T cells are eliminated by apoptosis
in a process known as negative selection
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– resulting in only around 2% of the immature T cells
reaching maturity.
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T-cells that leave the thymus (via the corticomedullarly junction) are singly positive, selfrestricted and self-tolerant.
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Thymus dysfunctions
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People without a fully functioning thymus can't
make enough T cells
– as a result, they are very vulnerable to infections.
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This is a particular problem for bone marrow
transplant patients
– Because a functioning thymus is needed to rebuild the
immune system once the transplant has been received.
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Some newborns also have malfunctioning or
completely absent thymus
– Due to conditions such as DiGeorge syndrome.
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Treatment
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Thymus disorders can sometimes be treated with
– Infusions of extra immune cells
– Transplantation of a thymus organ soon after birth
– But both are limited by a lack of donors and problems
matching tissue to the recipient.
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Being able to create a complete transplantable
thymus from cells in a lab would be a huge step
forward in treating such conditions.
Thymic involution
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Unlike most organs that grow until the age of
maturity
– the thymus enlarges throughout childhood
– but slowly shrinks from the onset of puberty and
throughout adulthood.
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As the thymus shrinks, its tissues are replaced by
adipose tissue. This process is called thymic
involution
The shrinking may be due to the reduced role of
the thyroid in adulthood or increased secretion of
sex steroids
The immune system produces most of its T cells
during childhood and requires very few new T cells
after puberty.
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Need for research
Thymus transplantations soon after birth can
increase adaptive immunity in patients who are
congenitally athymic
 But these transplantations are limited by donor
tissue supply and histocompatibility
 These limitations would be overcome if
functional thymic epithelial cells (TECs) could be
generated or expanded in vitro, i.e. they are able
to
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◦ produce mature T-cells
◦ produce well-formed organ with the same structure as
a healthy thymus
History of research

Several investigators have reported derivation of
TEC-like cells from pluripotent cells
1. Lai and Jin, (2009) and Lai et al., (2011) used mouse
embryonic stem cells to produce the cells that had
phenotype of thymic epithelial cells using FGF-7, FGF10 and BMP-4.
2. Inami et al., (2011) produced thymic epithelial
progenitor cells (TEPCs) by phenotype – by culturing
induced pluripotent stem cells (iPSCs) with collagen IV
coated dishes in the presence of activin A and lithium
chloride (LiCl) in mice
3. Parent et al., (2013) used human pluripotent stem cells
to generate thymic epithelial cells – by regulating
TGFβ, BMP4, RA,Wnt, Shh and FGF signaling
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Continued…
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Sun et al., (2013) produced TECs after
transplantation of the pluripotent cell-derived
TECs using a transcription factor autoimmune
regulator (AIRE)
 But in the experiment, scientists were unable to
produce cortical and medullary compartments of
thymus.
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In all these experiments
◦ Unable to produce TEC-like cells of an organized
thymus containing all TEC subtypes
◦ TEC-like cells had no capacity to support T-cell
development in vitro
Generation of TECs
Bredenkamp et al., 2014 from University of
Edinburgh carried out their study using cells
called fibroblasts taken from mouse embryos
(MEFs).
 The
transcription
factor
forkhead
box
N1 (FOXN1) is critically required for
development of thymic epithelial cells (TECs),
during embryonic development
 By increasing levels of the protein FOXN1,
scientists observed that the morphology of MEFs
had changed from fibroblasts’ cell shape to
epithelial cells shape.
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The experiment
Rosa26CreERt2/+ mice were taken as controls
 Transgenic mouse line was developed in
which
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◦ Foxn1 cDNA controlled by CAG promoter was knocked
into ROSA26 locus with a LoxP-flanked transcriptional
stop cassette was inserted between CAG promoter and
Foxn1 cDNA
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Continued…
These mice were then crossed with Rosa26CreERt2
generating Rosa26CreERt2/CAG-STOP-Foxn1-IRES-GFP embryos.
 From these embryos, primary mouse embryonic
fibroblast (MEFs) were generated
 These MEFs were then treated with tamoxifen to
remove
STOP
cassette
generating
Rosa26CreERt2/CAG-Foxn1-IRES-GFP iFoxn1 MEFs
 After 10 days of iFoxn1 expression initiation, the
morphology of cells changed from elongated,
bipolar shape characteristic of fibroblast cells to
broader, polygonal shape, characteristic of
epithelial cells
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Bright-field (left) and immunofluorescence images (right) showing
morphology and K8 staining, 10 days after 4OHT treatment.
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Checking the identity of cells
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The identity of these cells was further confirmed
by using
◦ Epithelial-specific markers keratin 8 (K8) and
◦ Epithelial cell adhesion molecule (EpCAM)
 These are expressed by all TECs during early thymus
development.
Most of iFoxn1 MEFs, but no control MEFs,
showed K8 expression
 Approximately 15% iFoxn1 MEFs expressed
EpCAM
 This suggested that FOXN1 induction had
converted the fibroblasts to an epithelial-like state.
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Continued…
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Further, EpCAM+ cells were isolated and analyzed
for expression of specific FOXN1-regulated genes
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TEC- (Dll4, Ccl25 and Kitl)
Cutaneous epithelium- (Fgf2 and Krt1)
Epithelial V-like antigen (Eva or Mpzl2)
non-Foxn1 target TEC-associated genes (Pax9 and Trp63)
The iFoxn1 MEFs but not control MEFs expressed
all genes except cutaneous epithelium genes.
 All these expressions suggested FOXN1-mediated
conversion of MEFs into TEC-like cells
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Functional attributes of iTECs
To test whether induced thymic epithelial cells
(iTECs) were functional or not, scientists
cultured a monolayer of iTECs with early T
lineage progenitors (ETPs) Lin-CD25-C-Kit+
 Analysis after 12 days of co-culture revealed the
presence of CD4+CD8+ double positive, CD4+
and CD8+ single positive cells resembling that of
adult mouse thymus
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Continued…
These T cells expressed both CD3 (role in antigen
recognition) and T-cell antigen receptor beta
 While, ETPs seeded onto control MEFs did not
enter thymopoiesis
 Interestingly, the capacity of iTECs to support
thymocyte development was dependent on their
plating density
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◦ high density (>500 cellsmm-2) producing >3 times more
CD4+ CD8+ T cells within 12 days than a lower density
(<250 cellsmm-2)
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iTECs can form functional thymus
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iTEC were then grafted under the kidney capsule of
genetically identical adult mice
Fetal thymic mesenchyme was included to ensure that
growth factors essential for expansion of the thymus,
including FGF10 and IGF, were available within the
graft
After four weeks, the cells had produced macroscopic,
well-formed organs with the same structure as a
healthy thymus, with clearly defined regions (known as
the cortex and medulla).
All EpCAM+ cells in the iTEC grafts expressed GFP,
reporting the transgenic iFoxn1-IRES-GFP messenger
RNA, confirming they were derived from the input
iTECs
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Hematoxylin and
eosin staining
reveals cortical and
medullary portions
of thymus
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Pan-cytokeratin
staining reveals
reticular network of
epithelial cells
throughout the
organs
Advantages
iTECs are a new and readily available source of TECs, that
may provide the basis for thymus transplantation therapies
aimed at boosting adaptive immune system function in
immuno-compromised patients.
 The technique may also offer a way of making patientmatched T cells in the laboratory that could be used in cell
therapies.
 Such treatments could benefit bone marrow transplant
patients, by helping speed up the rate at which they rebuild
their immune system after transplant.
 The discovery offers hope to babies born with genetic
conditions that prevent the thymus from developing
properly.
 Older people could also be helped as the thymus is the
first organ to deteriorate with age.
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Future enhancements
This is an exciting study but much more
work will be needed before this process can
be reproduced in a safe and tightly
controlled way suitable for use in humans
 With further refinements, the researchers
hope that their lab-grown cells could form
the basis of a thymus transplant treatment
for people with a weakened immune
system.
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Conclusion
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Enforced expression of FOXN1 is sufficient
to convert fibroblasts into iTECs
◦ an in vitro generated cell type that exhibits
phenotypic and functional properties of in vivo
TECs.
iTECs are able to promote full T-cell
development in vitro
 iTECs generate a properly patterned,
functional organ on transplantation in vivo,
composed of cortex and medulla of
thymus
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Reference
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Bredenkamp, N., S. Ulyanchenko, K. E.
O'Neill, N. R. Manley, H. J. Vaidya and C. C.
Blackburn. 2014. An organized and
functional thymus generated from FOXN1reprogrammed fibroblasts. Nat. Cell Biol.,
1-15.
Abbreviations used
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HSC = Hematopoietic
stem cells
MHC = Major
histocompatibility complex
FGF = Fibroblast growth
factor
BMP = Bone
morphogenetic protein
TECs = Thymic epithelial
cells
MEFs = Mouse embryonic
fibroblasts
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TGFβ = Transforming
growth factor β
RA = Retinoic acid
Wnt = Wingless type
Shh = Sonic hedgehog
IRES = Internal ribosome
entry site
GFP = Green fluorescent
protein
K8 = Keratin 8
EpCAM = Epithelial cell
adhesion molecule
Continued…
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4OHT = 4-hydroxy
tamoxifen
Dll4 = Delta like
ligand 4
Ccl25 = Chemokine
ligand 25
Kitl = Kit ligand
Krt1= Keratin 1
Mpzl2 = Myelin
protein zero-like 2
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Pax9 = Paired box gene
9
Trp63 = Transformation
related protein 63
CD3 = Cluster of
differentiation 3
IGF = Insulin like growth
factor
iTECs = Induced thymic
epithelial cells