University of Southern California Dissertations and Theses

IL-7R and c-Kit signaling in thymopoiesis

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IL-7R and c-Kit signaling in thymopoiesis

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IL-7R AND C-KIT SIGNALING IN THYMOPOIESIS

by

Akira Toyama

___________________________________________________________________

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CRANIOFACIAL BIOLOGY)

December 2008

Copyright 2008 Akira Toyama

ii
Table of Contents

List of Figures iii

Abstract iv

Chapter 1
Introduction – Thymic Development and Thymopoiesis 1
1.1 Thymus organogenesis 1
1.2 Thymic microenvironment 3
1.3 Thymocyte precursors 7
1.4 Signaling pathways involved in thymocyte development 9

Chapter 2
IL-7R and c-kit signaling has synergistic, essential and partially redundant
roles in thymopoiesis
2.1 Abstract 18
2.2 Introduction 19
2.3 Materials and Methods 21
2.4 Results 25
2.5 Discussion 36

Chapter 3
Determination of nature of IL-7R/c-kit signaling pathways - permissive or
instructive action – through transgenic expression of Bcl-2 in thymocytes
3.1 Abstract 41
3.2 Introduction 42
3.3 Materials and Methods 44
3.4 Results 45
3.5 Discussion 50

Chapter 4
Effects of IL-7R and c-kit signaling on thymic architecture
4.1 Abstract 54
4.2 Introduction 55
4.3 Materials and Methods 56
4.4 Results 57
4.5 Discussion 60

Conclusions and future directions 62

Alphabetized bibliography 65

iii
List of Figures

Figure 1.1 Ligand independent activation of IL-7R by activated c-kit 16

Figure 2.1 PCR genotyping of the mouse strains used for this study. 27

Figure 2.2 Synergistic effects of IL-7Rα
-/-
and Kit
W41/W41
mutations in 28
thymopoiesis

Figure 2.3 Blocked thymocyte differentiation in IL-7
-/-
Kit
W41/W41
and 29
IL-7Rα
-/-
Kit
W41/W41
mice.

Figure 2.4 Differentiation block in DN thymocytes of IL-7-/- Kit
W41/W41
and 30
IL-7Rα-/- Kit
W41/W41
mice.

Figure 2.5 Lack of TCR Dβ-Jβ gene rearrangement in IL-7
-/-
Kit
W41/W41
mice. 31

Figure 2.6 Lack of TCR Dβ-Jβ gene rearrangement in IL-7Rα
-/-
Kit
W41/W41
mice. 32

Figure 2.7 Reduced early T-lineage progenitor frequency in IL-7Rα
-/-
, 35
IL-7
-/-
Kit
W41/W41
, and IL-7Rα
-/-
Kit
W41/W41
mice.

Figure 3.1 Thymocyte numbers were significantly increased by the bcl-2 47
transgene in the presence of a functional c-kit.

Figure 3.2 DN thymocyte differentiation block is not relieved by enforced 48
expression of bcl-2 when a functional c-kit is absent.

Figure 3.3 Relieve of DN to DP differentiation block in IL-7Rα
−/−
Kit
W41/W41
mice 49
by enforced expression of bcl-2.

Figure 4.1 Lack of cortical and medullary regions in IL-7
-/-
Kit
W41/W41
and 58
IL-7Rα
-/-
Kit
W41/W41
mice.

Figure 4.2 TEC differentiation block in IL-7Ra
-/-
Kit
W41/W41
thymus and relieve 59
of the differentiation block by enforced bcl-2 expression.

iv
Abstract

IL-7 and Kit ligand (KL) are cytokines produced by thymic epithelial cells,
which interact with their cognate receptors on immature thymocytes. The IL-7R is
comprised of the IL-7Rα and common γ chain (γc) and has no intrinsic kinase
activity, while KL binds to the receptor tyrosine kinase Kit. Both IL-7Rα
-/-
and IL-7
-/-

mice have profound defects in thymopoiesis, although for unexplained reasons, the
defects in differentiation and thymic cellularity are more severe for IL-7Rα -/- than
IL-7-/- mice. In order to understand possible interactions between IL-7R and Kit
signaling in vivo, we generated doubly mutated mice which were homozygous for
the Kit
W41
loss of function mutation and null for either IL-7 or IL-7Rα. While IL-7
-/-
and IL-7Rα
-/-
mice had a 90-99% reduction in thymic cellularity and the Kit
W41/W41

mice had a 50% reduction, the IL-7
-/-
Kit
W41/W41
and IL-7Rα
-/-
Kit
W41/W41
mice had fewer
than 200 thymocytes, representing a 5-6 log decrease in thymic cellularity. The
thymocytes in the IL-7
-/-
Kit
W41/W41
and IL-7Rα
-/-
Kit
W41/W41
mice were blocked at the
earliest recognizable stage of thymic differentiation. The frequency of early T-
lineage progenitors (ETP) in IL-7Rα
-/-
, IL-7
-/-
Kit
W41/W41
, and IL-7Rα
-/-
Kit
W41/W41
mice
was significantly reduced compared to parental strains or wild type mice.
Introduction of a bcl-2 transgene did not relieve the block in differentiation of CD4-
CD8- (DN) thymocytes, or reduction in ETP absolute numbers in IL-7Rα
-/-
Kit
W41/W41

mice, but partially rescued IL-7Rα
-/-
mice. Cytokeratin expression analysis showed
that thymic epithelial cells (TEC) of IL-7
-/-
Kit
W41/W41
, and IL-7Rα
-/-
Kit
W41/W41
mice were
K8+K5+, indicating that differentiation of TEC was arrested in these mice. IL-7Rα
-/-
Kit
W41/W41
transgenic bcl-2 thymuses had K8+K5- areas indicating that medullary
areas developed. Conclusions: 1) IL-7R and Kit provide synergistic, partially

v
redundant, and unique signals for thymocyte proliferation, maintenance, and
differentiation; 2) the less severe defect in IL-7
-/-
mice is due to partial
complementation by Kit, possibly by direct interaction between IL-7R and Kit; 3) a
functional Kit pathway is required in order for the bcl-2 transgene to partially rescue
IL-7Rα
-/-
mice; 4) although ETP do not express IL-7R, they are dependent on IL-7R
signaling for generation.

1
Chapter 1

Introduction - Thymic development and Thymopoiesis

The thymus is a complex structure which consists of a mesenchyme-derived
capsule surrounding a network of stromal components, which include endoderm
derived epithelial cells, hematopoietic-derived thymocytes, macrophages, dendritic
cells and blood vessels. The thymus is the primary lymphoid organ crucial for
functional T cell development and a properly functioning immune response. T cell
progenitors generated in fetal liver or bone marrow are recruited to and colonize the
thymus via blood (Katsura 2002, Marman 2005, Masuda 2005) and proliferate and
differentiate within the thymic microenvironment which is created by stromal cells
(Itoi 2007). Commitment of precursors to the T cell lineage and T cell repertoire
selection also occur in the thymus (Itoi 2007). These unique functions of the thymus
are mediated primarily through interactions between developing thymocytes and the
microenvironments which are mainly defined by the thymic epithelial components of
the stroma (Osada 2006).
The major component of the thymic stroma is epithelial cells which generate
the three-dimensional meshwork structure unique to the thymus (van Ewijk 1999).
Thymic epithelial cells provide various signals including cytokines that are essential
for T cell development. (Itoi 2007) This chapter will review (thymic anlage and
progenitors, function, microenvironment, and thymocyte progenitors) of the thymus.

1.1 Thymus anlage and thymocyte progenitors
Initiation of thymus development is thought to occur at approximately E9.5
through epithelial–mesenchymal interactions in the region of the third pharyngeal

2
pouch. The first identifiable morphological signs of thymus development become
apparent by embryonic day (E) 10.5 (Manley 2000). Epithelial cells of the mouse
thymus anlage originate in the third pharyngeal pouch endoderm (Bennett 2002),
which protrudes into the pharyngeal arch mesenchymal region on E 9-11 (Bennett
2002). This results in formation of the thymus anlage (Cordier 1980, Manley 2000).
Epithelial cells of the anlage on E11 show a stratified bilayer structure, and a
meshwork structure on E13 and thereafter (Itoi 2001). From the early stage of
thymus anlage development, epithelial cells express functional molecules essential
for early T cell development including CCL21, CCL25 (Liu 2005, Bleul 2000), IL-7
(Zamisch 2005), and delta like (Dll) 1 and Dll4 (Harman 2003). Initial colonization of
T cell progenitors to the thymus anlage occurs around E11 (Itoi 2001). The earliest
T lineage progenitors migrate among thymic epithelial cells and immediately start
proliferation and differentiation in the anlage on E12 (Amagai 1995).
Epithelial-mesenchymal interactions have essential roles in thymus
organogenesis. Roles of mesenchymal cells in functional development of epithelial
cells in the thymus anlage was examined in patch (ph) mutant mice, which have a
primarily defect in mesenchymal cells caused by the absence of platelet-derived
growth factor receptor a expression (Itoi 2007). In the ph/ph thymus anlage, T cell
progenitors migrate normally among the epithelial cells, however, they are severely
impaired to proliferate and differentiate to CD25-positive CD4-CD8- double negative
(DN) DN2 and DN3 cells. Epithelial cells of the ph/ph thymus anlage exhibit severely
impaired proliferation and expression of functional molecules, such as SCF, Delta-
like 4 and MHC class II, which have crucial roles in T cell development. Furthermore,

3
the cultured ph/ph thymus anlage fails to develop into a mature thyus which can
support functional T cell development (Itoi 2007).

Studies show that mesenchymal cells contribute to development of epithelial
cells of the thymus anlage. For example, lobule formation and outgrowth of epithelial
cells of the early thymus anlage require interaction with mesenchymal cells
(Auerbach 1960, Shinohara 1996). Mesenchymal cells of the thymus anlage
produce fibroblast growth factor (FGF) 7 and FGF10. These FGFs induce
proliferation of epithelial cells through their receptor FGFR2IIIb indicating that the
proliferation of epithelial cells of the thymus anlage depends on interaction with
mesenchymal cells (Ohuchi 2000, Revest 2001). It has been shown that
mesenchymal cells are of key importance for the thymus anlage to develop into a
functionally mature organ supporting full T cell development (Itoi 1998). In addition,
mesenchymal cells have been reported to induce expression of MHC class II
molecules on thymic epithelial cells (Itoi 1998, Shinohara 1997). These observations
suggest that mesenchymal cells have a crucial role not only in proliferation but also
in differentiation of thymic epithelial cells (Itoi 2007).

1.2 Thymic structure and microenvironment
Thymus is organized into cortical and medullary areas, and thymic epithelial
cells in the two regions have specific function in thymocyte development. Thymus
seeding cells which have a high T-lineage cell generation capacity enter the thymus
via blood and mature into functional T cells. Thymic epithelial cells (TEC) play
essential roles in this process. Cortical TECs support early T cell progenitor

4
commitment and differentiation and play a central role in the processes of β -
selection and positive selection (Anderson 1993, 1994). In contrast, medullary TECs,
in conjunction with dendritic cells, are essential for effective negative selection
during the late stages of thymocyte development (Derbinski 2001, Gallegos 2004).
Differentiation of T lineage progenitors which seed the thymus can be
classified based on the expression of CD4 and CD8. Immature thymocytes are
doubly negative for both CD4 and CD8 (double negative, DN). DN cells are further
divided into four differentiation stages based on the expression of CD25 and CD44
(Godfrey 1992). DN1: CD44+CD25-, DN2, CD44+CD25+, DN3, CD44-CD25+, DN4,
CD44-CD25-. DN4 cells further differentiate into CD4 CD8 double positive cells
which then differentiate into either CD4+ or CD8+ single positive (SP) cells. SP cells
are finally exported to the periphery as functional T cells.
Petrie and Zuniga-Pflucker (2007) classified regions of the thymus according
to their functional roles in thymopoiesis. The following subsections will review the
role of each thymic region in thymocyte development.

Perimedullary cortex.
The perimedullary cortex where signaling to DN1 cells occurs, is defined by
by the location of DN1 cells (CD25− CD44+ CD117+), spanning a very narrow
region of the cortex adjacent to the medulla (Lind 2001, Porritt 2003). The mean
time of DN1 cell residence in this zone is ten days (Porritt 2003, Shortman 1990).
This is where recruitment of marrow-derived progenitors from the blood, proliferative
expansion of the DN1 pool, retention and asynchronous release of DN1 cells as an
intrathymic progenitor cell pool, and T-lineage specification occur. Known or

5
predicted stromal signals provided in this region include: fibronectin, laminin, VCAM-
1, MadCAM-1, ICAM-1, P-selectin, CXCL12, and CCL21 (homing and
extravasation), Kit ligand, IL-7, Hedgehog (proliferation, survival, and/or adhesion),
and Notch ligands (T lineage specification).

Inner cortex.
Inner cortex where signaling to DN2 cells (CD25+ CD44+ CD117+) occurs,
is loosely defined as spanning the inner half of the cortex, excluding premedullary
cortex. The mean time of DN2 cell residence is approximately two days (Porritt 2003,
Shortman 1990, Penit 1995). This is where induction of polarized migration toward
the capsule, proliferative expansion, induction of recombinase activity and TCRγ/δ
recombination, lineage divergence (TCRα/β versus TCRγ/δ), and continued T
lineage specification occur. Known or predicted stromal signals provided in this
region include: CXCL12, CCL25, VCAM-1, E-cadherin (adhesion/migration), IL-7,
Kit ligand (involved in survival/proliferation), and Notch ligands (lineage
specification).

Outer cortex.
Outer cortex where signaling to early DN3 cells (CD25+ CD27lo CD44lo
CD117lo) occurs, is loosely defined as spanning the outer half of the cortex (Porritt
2003). Mean time of DN3 cell residence is two days (Porritt 2003, Shortman 1990,
Penit 1995). This is where migration toward the capsule, proliferative expansion,
TCRβ recombination and TCRα/β versus TCRγ/δ lineage divergence, and absolute
T lineage commitment occur. Known or predicted stromal signals provided in this

6
region include: VCAM-1 (migration), IL-7 (survival), Hedgehog (proliferation), and
Notch ligands (T lineage specification).

Subcapsular zone.
Subcapsular zone where signaling to late DN3 (CD24+ CD25+ CD27hi
CD44lo CD117−) and pre-DP (CD4
lo
CD8
lo
CD25− CD44
lo
) cells occurs, is the area
immediately adjacent to the capsule, defined by a dense band of proliferating cells
(Penit 1988, Lind 2001). Mean time of lymphoid cell residence is roughly one day or
less. This is where the completion of TCRβ rearrangements, pre-TCR expression
and TCRβ selection, cell death of nonselected cells, acquisition of CD4 and CD8
expression, reversal of polarity of migration, and induction of TCRα recombination
occur. Known or predicted stromal signals provided in this region include: CCL25,
laminin-5 (migration), CD70 (survival), Notch ligands (survival, T lineage
specification).

Cortex, moving inward.
Cortex, where signaling to DP cells occurs, is all of the cortex excluding the
subcapsular zone. The mean time of lymphoid residence is 1.5 to 2 days (Penit
1988, Penit 1995, Egerton 1990). This is where cell cycle withdrawal, ongoing
TCRα recombination, semi-random migration, positive selection, and CD4/CD8
lineage divergence occur. Known or predicted stromal signals provided in this region
include: IL-7 (survival, lineage divergence), MHC, ICAM-1 (positive selection), and
CCL25 (polarized migration).

7
Outer medulla.
Outer medulla, where signaling to postpositive selection CD4 or CD8 SP
cells (also characterized as CD24+ CD62L
lo
CD69+ Qa-2−) occurs, is the outer
regions of the medulla, defined by the presence of a high density of DCs. Mean time
of lymphoid residence in this region is unclear. The mean total time of medullary
residence is 5–7 days (Egerton 1990), and cells may encounter this zone more than
once during that time. This is where chemoattraction of positively selected cells from
the cortex and negative selection occur. Known or predicted stromal signals
provided in this region include: MHC, CD80, CD86, ICAM-1 (negative selection), IL-
7 and TSLP (survival), CCL17, CCL19, and CCL21 (chemoattraction).

Central medulla.
Central medulla, where signaling to nearly mature CD4 or CD8 SP cells
(also characterized as CD24
lo
CD62L
hi
CD69− Qa-2+) occurs, is a poorly defined
region in the medulla outside of the DC-rich outer region. Mean time of lymphoid
residence in this region is 7–10 total days in the medulla, including outer medulla
(Egerton 1990, Gabor 1997). This is where retention, tolerance induction,
endowment of functional capacity, and exportation from the thymus occur. Known or
predicted stromal signals provided in this region include: MHC, LTβR (functional
maturation), CD69 (retention), CCL12, S1P-S1P1 (thymic egress).

1.3 Thymocyte precursors
A. Putative thymus seeding precursors
The nature of thymus seeding precursors has not been determined to date.

8
hematopoietic stem cells, multipotent progenitors, early lymphoid progenitors
(lymphoid-specified), common lymphoid progenitors, and circulating T cell
progenitors (Bhandoola 2007). While a number of potential T cell progenitors have
been identified, the relative contribution of multiple progenitors to T cell production
has not been determined. No one progenitor has been identified as the predominant
physiological T lin progenitor. There is evidence which suggests canonical T lineage
progenitors may not be physiologically important in (Umland 2007).

B. Importation of precursors into thymus
Development of thymocytes critically depends on availability of thymic niche
for progenitor cells that seed the thymus. A number of studies have demonstrated
that intrathymic binding sites and microvascular gates exist that control entry and
seeding of progenitors into the thymus (Foss 2002, Goldschneider 2006, Donskoy
2003, Foss 2001). Intrathymic binding sites are likely to be associated with cell
adhesion molecules and chemokines (Schwartz 2006, Zubkova 2005). Intrathymic
microvascular gates and availability of specific intrathymic microenvironmental
niches appear to regulate progenitor importation into the thymus (Foss 2002). Also
cyclic nature of this importation of progenitors has been observed (Foss 2002,
Goldschneider 2006, Donskoy 2003).

C. Precursors within the thymus
The nature of thymus seeding T lineage progenitors remains to be
elucidated. This is mainly due to the expected small number of such progenitors
which seed the thymus periodically and associated difficulty in identifying the

9
progenitors. Potential T lineage progenitors in the thymus include LSK (lin- Sca-1+
c-kit+), DN1a and DN1b (CD44+ CD25- CD24+/-), common lymphoid progenitors
(Lin- Sca-1
lo
IL-7Rα+ c-kit
lo
; Flt3+) and early T lineage progenitors (lin- CD44
hi

Sca-1+ IL-7Rα
-/lo
c-kit
hi
).

1.4 Signaling pathways involved in thymocyte development
A. IL-7/IL-7R signaling pathways
IL-7 exerts its biological activities by signaling through a receptor complex
consisting of the IL-7Rα chain and the cytokine receptor common gamma chain, γc.
Both of these chains are required for functional responses to IL-7 (DiSanto 1995,
Leonhard 1995). IL-7 is expressed in bone marrow and thymus stromal cells, and
target cells of IL-7 include lymphoid precursors that express IL-7Rα (Sudo 1993,
Era 1994) and γc (Kondo 1994, Orlic 1997). Downstream pathways involved in IL-
7R signaling include STAT5, PI3K-Akt-mTOR, and Ras-MAPK, and proliferation
(through c-Myc, NF-ΚB, and Cyclin D) and differentiation (through TCRγ, Bcl-2, and
CIS1) of IL-7R expressing cells occur (Kang 2004). The in vivo roles of IL-7, IL-7Rα
and γc have been analyzed in mouse mutants lacking each of these molecules and
are reviewed below.

B. IL-7-/- mice
Genetic deletion of IL-7 in mice clearly defined the role of IL-7 in
thymopoiesis in vivo (von Freeden-Jeffry 1995). IL-7-/- mice showed a severe
reduction in the number of pro-B and pro-T cells and a marked inhibition of the

10
differentiation of these cells to subsequent stages. There is an incomplete
intrathymic differentiation block at the transition from the CD44+CD25+ (DN2) to the
CD44-CD25+ (DN3) stage (Moore 1996), where active proliferation of T-cell
precursors occur in wild type mice (O’Reilly 1997, Penit 1995). Subsequent to the
CD44+CD25+ stage, maturation of pro-T cells that have developed to this stage is
possible in the absence of IL-7; in contrast, development of γδ T cells is abrogated
(Moore 1996). These results indicate that IL-7 is important for the survival and/or
proliferation of early lymphoid precursors and essential for the development of γδ T
cells.

C. IL-7Rα-/- mice
Similar to IL-7-/- mice, IL-7Rα-/- mice have hypoplastic thymuses and lack γδ
T cells (Maki 1996, He 1996). In one IL-7Rα-/- strain (Maraskovsky 1997), a large
fraction (65%) of mice appear to have a more complete block in thymocyte
differentiation (between DN to DP stages) than IL-7-/- mice. The thymuses of these
mice are composed entirely of double negative (DN) cells at DN1 (CD44+CD25-). In
contrast, the remaining 35% of mice have an overall thymic development
resembling those of IL-7-/- or γc- mice (i.e. the presence of the four major CD4/CD8
subsets with an overall 20-fold thymic cellularity reduction). The mechanism
responsible for this thymocyte phenotypic variability within this mouse strain remains
undefined. An independently derived line of IL-7Rα
-/-
mutant mice show a thymic
phenotype that closely resembles γc- or IL-7
-/-
mice - permissive T-cell development
at reduced cellularity - in 100% of mice examined (Maki 1996).

11
D. Common cytokine receptor γc-deficient mice
All the features of IL-7 deficiency, including severe lymphopenia and the
absence of γδ T cells, are also found in γc- mice (Cao 1995). γc- mice exhibit a
similar incomplete intrathymic differentiation block at the CD44+CD25+ stage as IL-
7-/- mice (Moore 1996 ,DiSanto 1995, Ohbo 1996), suggesting that other γc-
dependent cytokines (IL-2, IL-4, IL-9, IL-15, and IL-21, Sugamura 1995) do not
additionally intervene at this stage of early thymocyte development in the thymus.
γc-deficient mice demonstrate differences from IL-7-/- mice in peripheral immune
phenotype, such as a complete absence of NK cells, NK-T cells and gut-associated
lymphoid tissue due to abrogation of IL-15 signaling, which is critical for these
functions.

E. SCF/c-kit
Germline mutations in c-kit which is encoded at the W locus or its ligand
stem cell factor, SCF, which is encoded at the S1 locus, affect the development of
several cell lineages including melanocytes, germ cells, and hematopoietic cells (Di
Santo and Rodewald, 1998). Signaling through c-kit/SCF regulates the development,
maintenance or function of these lineages. As a result, the lack of c-kit or of SCF
proteins can cause cutaneous white spots, sterility, altered gut motility and
hematopoietic defects. c-kit is expressed in very early hematopoietic cells including
hematopoietic stem cells (HSC). SCF is provided in either soluble or membrane-
bound form by microenvironments that support the development or function of c-kit
expressing lineages. Mice homozygous for c-kit null mutations (W/W) die
approximately 10 days after birth due to severe anemia (Russell 1979).

12
Homozygous loss of SCF (SI/S1) is lethal in late gestation in mice (Galli 1994). In
the T-cell lineage, c-kit is expressed in subsets of very early fetal and adult
intrathymic precursors (Shortman 1996, Wu 1991). CD44+CD25- (DN1) and
CD44+CD25 + (DN2) thymocytes express c-kit but expression is lost in CD44-CD25
+ (DN3) thymocytes (Moore 1995). Thus, c-kit appears to be expressed for as long
as T cell progenitors lack T cell receptor V(D)J chain rearrangements (Di Santo and
Rodewald, 1998).

F. Alternative pathways/cells
There are alternative pathways that allow T lineage differentiation in the
absence of canonical T cell progenitors. Recently, involvement of fms-like tyrosine
kinase 3 ligand (Flt3l) and thymic stromal lymphopoietin (TSLP) in thymopoiesis has
been elucidated (Sitnicka 2007, Chappaz 2007). Flt3l has been shown to be
important in IL-7R-independent thymopoiesis (Sitnicka 2007). Flt3l-/-IL-7R-/- mice
have extensive reductions in fetal and postnatal thymic progenitors and a loss of
thymopoiesis indicating that Flt3l has an indispensable role in IL-7R independent
thymopoiesis (Sitnicka 2007). Transgenic expression of TSLP in adult IL-7-/- mice
resulted in restoration of T and B cell compartments (Chappaz 2007). In vitro
experiments have shown that TSLP promoted the differentiation of uncommitted
adult bone marrow progenitors toward B and T lineages (Chappaz 2007).
Differentiation of DN1 and DN2 thymocytes was also induced by transgenic TSLP
expression (Chappaz 2007).
Studies of IL-7-/- (von Freeden-Jeffry 1995), IL-7Rα-/- (Peschon 1994), and
various Kit mutant strains with partial activity including Kit
W41/W41
(Nocka 1990)

13
mutant mice revealed importance of IL-7R and c-kit pathways in thymocyte
proliferation and differentiation. While Kit
W41/W41
mice have approximately 50% of
normal thymocyte numbers, IL-7-/- and IL-7Rα-/- have 2-3% or less than 1% of
normal, respectively. There is no detectable block in differentiation of Kit
W/W

thymocytes even though thymocyte proliferation was reduced 40-fold at DN stage
(Rodewald 1995). However, IL-7-/- and IL-7Rα-/- thymocytes have a block in
differentiation at CD4-CD8- (double negative, DN) stage 3 and 2, respectively
(Peschon 1994, von Freeden-Jeffry 1995).

G. Combined IL-7R and Kit
W41/W41
mutations
In order to examine the effect of combined IL-7 or IL-7R and Kit
W41/W41

mutations in thymopoiesis, IL-7-/- Kit
W41/W41
and IL-7Rα-/- Kit
W41/W41
double mutant
mice were generated and the mice were examined for their thymocyte cellularity and
differentiation and thymus histology. Kit
W41/W41
mice were chosen to be used in this
study because these mice are viable and fertile whereas Kit
W/W
mice die neonatally.
The focus of this thesis is on elucidating the effect and possible mechanism of the
combined IL-7 or IL-7Rα and Kit
W41/W41
mutations in thymopoiesis.

H. Ligand-independent activation of cytokine receptors
Signaling of cytokine receptors involved in thympoiesis may be induced by
ligation of alternative ligands (i.e. TSLP) or mediated by alternative cytokine
signaling pathway (i.e. Flt3). Contribution of ligand-independent activation of
cytokine receptors in differentiation and proliferation of hematopoietic cells has been

14
elucidated. For example, ligand-independent activation of erythropoietin receptor
(EpoR) by activated c-kit has been shown to contribute to erythropoiesis (Wu et al.
1995). The c-kit/Epo-R crosstalk involves direct interaction between c-kit and Epo-R
leading to tyrosine phosphorylation of Epo-R by c-kit in the absence of Epo.
c-kit has also been shown to interact with other type I cytokine receptors
such as IL-3R (Liu 1994). In addition, it has been shown that IL-2Rβγ signaling is
activated by c-kit in the absence of IL-2 in human papillomavirus-associated cervical
cancer. (Rocha-Zavaleta 2004). These studies show that it is possible for some
cytokine receptors to activate signaling pathways downstream of the receptors
without the ligation of their respective ligands.
In a recent study from the Weinberg laboratory (Jahn 2007), direct
interaction and activation of IL-7R by c-kit in the absence of SCF has been
elucidated. In vivo analyses of thymopoiesis in mice defective in signaling through c-
kit and γc or c-kit and IL-7Rα demonstrate synergy and partial complementation of
γc or IL-7– mediated signaling by the Kit signaling pathway (Rodewald 1997).
Molecular analysis in T lymphoid cells as well as in cells which are not of
hematopoietic origin shows that c-kit and IL-7R signaling pathways directly interact.
SCF mediated activation of c-kit induced strong tyrosine phosphorylation of γc and
IL-7Rα in the absence of IL-7. It was shown that activated c-kit formed a complex
with either IL-7Rα or γc, and tyrosine phosphorylation of both subunits occurred
independently of Jak3, suggesting that γc and IL-7Rα are each direct substrates of
activated c-kit. Based on the data, c-kit-mediated functional activation of γc-
containing receptors such as IL-7R, similar to the interaction between c-kit and Epo-
R is proposed in the study. Such indirect activation of the Jak-Stat pathway induced

15
by the interaction between a receptor tyrosine kinase such as c-kit and type I
cytokine receptor such as IL-7R could be responsible for the phenotypic difference
between IL-7-/- and IL-7Rα-/- mice (Jahn 2007). (Figure 1.1) This ligand-
independent activation of IL-7R by activated c-kit can occur in IL-7-/- but not in IL-
7Rα-/- thymocytes, and this may account for the phenotypic differences in thymuses
of the mutant mice.

I. Alternative ligands which support thymopoiesis
Less severe thymic phenotype of IL-7-/- mice (2-3% of normal thymocyte
number) compared to IL-7Rα-/- (less than 1% of wild type thymocyte number) may
be due to partial complementation of loss of IL-7R signaling by c-kit in IL-7-/-
thymocytes (Jahn et al. 2007). Alternatively, other ligands which can bind to the IL-
7R may account for the phenotypic differences between IL-7-/- and IL-7Rα-/- mice.
Recently it was shown that TSLP whose receptor (TSLPR) is comprised of IL-7Rα
and TSLP-specific TSLPR chain, induces proliferation of thymocytes (Al-Shami
2004, Chappaz 2007, Jiang 2007).
While TSLPR-/- mice had no defect in T development, mice lacking both the
γc and the TSLPR chains showed lower thymic cellularity than γc
-/-
mice indicating
that TSLP signaling contributes to T cell development (Al Shami 2004). Moreover,
the injection of recombinant TSLP could transiently increase the number of
thymocytes in γc
-/-
mice. (Chappaz 2007). A study by Chappaz and others has
shown that a transgenic expression of TSLP in IL-7
-/-
mice restores T cell

16

JAK3
IL-7Rα
KL
c-kit
JAK1
IL-7
γc
IL-7
-/-

IL-7R
Kit
Figure 1.1 Ligand independent activation of IL-7R by activated c-kit.
In the absence of IL-7, IL-7R may be activated by c-kit. This may explain
the phenotypic difference between IL-7-/- and IL-7Rα-/- mice because this
ligand-independent activation of IL-7R by c-kit can occur in IL-7-/-
thymocytes but not in IL-7Rα-/- thymocytes.

17
compartment – increased thymic and splenic cellularities, and restored double
negative (DN) thymocytes, αβ and γδ T-cell generation, and peripheral lymphoid
compartments (Chappaz 2007). This indicates that in the absence of IL-7R signaling,
signals downstream of TSLPR may partially complement IL-7R mediated signaling
in thymocytes.
Flt3 (Fms-like tyrosine kinase 3) has recently been shown to have significant
roles in IL-7R independent thymopoiesis (Sitnicka 2007). Flt3 ligand–deficient mice
have distinct reductions in the earliest thymic progenitors in fetal, postnatal, and
adult thymus. A critical role of Flt3 signaling in thymopoiesis becomes evident in the
absence of interleukin-7 receptor α (IL-7Rα) signaling. Flt3l-/-IL-7r-/- mice have
extensive reductions in fetal and postnatal thymic progenitors that result in a loss of
thymopoiesis in adult mice, demonstrating an indispensable role of Flt3L in IL-7Rα-
independent fetal and adult T lymphopoiesis (Sitnicka 2007). In addition, a unique
and critical role of Flt3L, distinct from that of IL-7Rα, in regulation of the earliest Lin-
Sca1+ KIT+ (LSK) Flt3hi lymphoid primed multipotent progenitors in the bone
marrow is elucidated in the same study. This demonstrates a key role of Flt3
signaling in regulating the very earliest stages of lymphoid progenitors. Because Flt3
pathway is intact in both IL-7-/-Kit
W41/W41
and IL-7Rα-/-Kit
W41/W41
mice, Flt3 signaling
may be responsible for the generation of residual thymocytes and lymphocytes in
these mice.

18
Chapter 2

IL-7R and c-kit signaling has synergistic, essential and partially redundant
roles in thymopoiesis.

2.1 Abstract

IL-7 and Kit ligand (KL) are cytokines produced by thymic epithelial cells,
which interact with their cognate receptors on immature thymocytes. The IL-7R is
comprised of the IL-7Rα and common γ chain (γc) and has no intrinsic kinase
activity, while KL binds to the receptor tyrosine kinase Kit. Both IL-7Rα
-/-
and IL-7
-/-

mice have profound defects in thymopoiesis, although for unexplained reasons, the
defects in differentiation and thymic cellularity are more severe for IL-7Rα -/- than
IL-7-/- mice. In order to understand possible interactions between IL-7R and Kit
signaling in vivo, we generated doubly mutated mice which were homozygous for
the Kit
W41
loss of function mutation and null for either IL-7 or IL-7Rα, and. While IL-
7
-/-
and IL-7Rα
-/-
mice had a 90-99% reduction in thymic cellularity and the Kit
W41/W41

mice had a 50% reduction, the IL-7
-/-
Kit
W41/W41
and IL-7Rα
-/-
Kit
W41/W41
mice had fewer
than 200 thymocytes, representing a 5-6 log decrease in thymic cellularity. The
thymocytes in the IL-7
-/-
Kit
W41/W41
and IL-7Rα
-/-
Kit
W41/W41
mice were blocked at the
earliest recognizable stage of thymic differentiation at DN1. The frequency of early
T-lineage progenitors (ETP) in IL-7Rα
-/-
, IL-7
-/-
Kit
W41/W41
, and IL-7Rα
-/-
Kit
W41/W41
mice
was significantly reduced compared to parental strains or wild type mice. From the
results of this study, the following conclusions were drawn: 1) IL-7R and Kit provide
synergistic, partially redundant, and unique signals for thymocyte proliferation,

19
maintenance, and differentiation; and 2) the less severe defect in IL-7 -/- mice is due
to partial complementation by Kit, possibly by direct interaction between IL-7R and
Kit.

2.2 Introduction
Two cytokines, interleukin 7 (IL-7) and c-kit ligand (KL), have been identified
as necessary for the normal proliferation and survival of pro-thymocytes. IL-7 has
been shown to have proliferative, differentiative and anti-apoptotic effects on
developing thymocytes. IL-7 binds to an IL-7R comprised of a ligand-specific IL-
7Rα subunit and the gamma common subunit (γc), which is also a subunit of the
receptors for IL-2, IL-4, IL-9, IL-15, and IL-21. Engagement of the IL-7R results in
activation of the JAK-STAT, PI3K-Akt, Ras-Erk, and other pathways. The signals
transduced by the IL-7R are critical for thymic differentiation. C-kit is a receptor
tyrosine kinase which is expressed in hematopoietic stem cells, committed
hematopoietic progenitors, melanocytes and germ cells. Both c-kit and kit ligand,
KL, have been shown to be important to thymopoiesis as c-kit or KL null mice have
reduced numbers of thymocytes. However, the defect in both c-kit and KL mutant
mice is primarily numerical with normal differentiation of thymocytes.
IL-7
-/-
mice have a less severe defect in thymopoiesis than that observed for
IL-7Rα
-/-
mice. IL-7
-/-
mice have 2-3% of normal thymus cellularity, and a partial
inhibition of differentiation of prothymocytes. IL-7Rα
-/-
mice have 1% of normal
thymus cellularity and an earlier block in differentiation of prothymocytes and
absence of TCR γδ cells.

20
The differences in the thymic phenotype between IL-7
-/-
and IL-7Rα
-/-
mice
suggest that an alternative ligand for the IL-7Rα which might partially complement
the defect in IL-7
-/-
mice. Alternatively, IL-7Rα might be a substrate for another
receptor such as c-kit which might be able to activate IL-7Rα in the absence of IL-7,
resulting in partial compensation of IL-7-/- phenotype. Biochemical and genetic
experiments in our lab and others have shown that the IL-7R and c-kit directly
interact, suggesting that the intact c-kit pathway may complement the loss of IL-7R
in IL-7Rα-/- transgenic bcl-2 mice.
In recent years a number of putative thymocyte progenitors have been
identified. Identification of common lymphoid progenitor (CLP) which can
differentiate into both T and B cells provided insight into the process of lymphoid
progenitor differentiation (Kondo 1997). A number of other putative thymocyte
progenitors were characterized, including LSK (lin- Sca-1+ c-kit+), DN1a and DN1b
(CD44+ CD25- CD24+/-), and early T-lineage progenitors (lin- CD44hi Sca-1+ IL-
7Rα-/lo c-kithi). However, relative physiological contribution of these possible
progenitors has not been determined. A recent study has shown that thymus
seeding T-lineage progenitors go through ETP stage, suggesting that ETP may
have important physiological roles in thymopoiesis (Benz 2008). In this chapter of
this study, frequency and absolute numbers of early T-lineage progenitors in mutant
as well as control mice were determined in order to determine the functional roles of
IL-7R and c-kit signaling pathways at this stage of thymocyte differentiation.

21
2.3 Materials and Methods

Animals.
The generation of the IL-7
-/-
and IL-7Rα
-/-
mice has previously been
described (von Freeden-Jeffry 1995, Peschon 1994). The IL-7
-/-
mice were back-
crossed at least 10 generations onto a C57BL/6 background. The IL-7Rα
-/-
,
Kit
W41/W41
, and Eµ-bcl-2-36 mice on a C57BL/6J background were purchased from
the Jackson Laboratories (Bar Harbor, ME). The mice were housed in the animal
care facility at Childrens Hospital Los Angeles in micro isolator cages. They were
fed regular chow and sterile water ad libitum. All experiments were performed under
protocols approved by the Institutional Animal Care and Use Committee of
Childrens Hospital Los Angeles.

Breeding.
To generate the IL-7
-/-
Kit
W41/W41
mice, mice heterozygous for both the IL-7
and c-kit alleles were generated by mating IL-7
-/-
mice with Kit
W41/W41
mice. A pair of
the IL-7
wt/-
Kit
wt/W41
mice were bred to generate male and female IL-7
-/-
Kit
W41/W41
mice.
Homozygous IL-7
-/-
Kit
W41/W41
mice were distinguished from IL-7
wt/-
littermates by
PCR as previously described (von Freeden-Jeffry 1995).

Eµ-bcl-2-36 transgenic
mice were bred with IL-7
-/-
, IL-7
-/-
Kit
W41/W41
, IL-7Rα
-/-
or IL-7Rα
-/-
Kit
W41/W41
mice to
generate IL-7
-/-
, IL-7
-/-
Kit
W41/W41
, IL-7Rα
-/-
or IL-7Rα
-/-
Kit
W41/W41
transgenic bcl-2 mice.
Genotyping of mice for expression of the transgene was performed by PCR using
primers A: 5’-CTA GGC CAC AGA ATT GAA AGA TCT-3’, B: 5’-GTA GGT GGA

22
AAT TCT AGC ATC ATC C-3’, C: 5’-TGG ATC CAG GAT AAC GGA AA-3’, and D:
5’-TGT TGA CTT CAC TTG TGG CC-3’ (Jackson Laboratory, Bar Habor, ME).
Primers A and B produce a 315bp band which is used as a loading control, and
primers C and D produce a 170bp band if a mouse possesses the bcl-2 transgene.
Molecular analysis of Kit
W41/W41
mutation was performed by restriction
analysis of a PCR product containing the Kit
W41
mutation in exon 18. Total RNA was
isolated from thymuses or bone marrow by using RNAgent total RNA isolation
system (Promega, Madision, WI), reverse transcribed with AMV-RT (Gibco BRL,
Gaithersburg, MD) and amplified by PCR. The segment of the c-Kit gene which
contains the W41 mutation (exon 18) was amplified with the sense primer 5’-ACA
AGA GGA GAT CCG CAA GA-3’ and antisense primer 5’-GGT AGG GGC TGC
TTC CTA AG-3’. PCR was done for 35 cycles (30 sec. at 94°C, 1 min at 60°C, 1 min
at 72°C). The PCR products were then digested with Xcm I (New England Biolab,
Beverly, MA). The Kit
W41
mutation at position 2519 (G→A) of the c-kit cDNA adds an
Xcm I restriction site, resulting in PCR products of 293 bp and 109 bp, instead of the
402 bp product resulting from amplification of the wild-type cDNA sequence.
IL-7Rα
-/-
Kit
W41/W41
mice were generated using a similar procedure as described for
generation of IL-7
-/-
Kit
W41/W41
mice. The IL-7Rα

null mutation was detected by PCR
with an antisence primer A: 5’ CTT TTA CGA GTG AAA TGC CTA ACT C 3’ and
sense primers B: 5’ CAG GTA TTC AAG AAT GCA ATA CA 3’, and C: 5’ CAC GGC
TAG CCA ACG CTA TGT C 3’ (Jackson Laboratory, Bar Habor, ME) were used to
identify the IL-7Rα
-/-
Kit
W41/W41
mice. Primers A and B amplify a 850 bp band from the

23
wild type allele, and primers A and C amplify a 950 bp band from the IL-7Rα

targeted allele.

Detection of T cell receptor β chain (D-J) rearrangement by PCR.
PCR-based analysis of TCR β chain rearrangement was performed as
previously described.
20
For detection of specific rearrangements of the Dβ – Jβ
gene segments of the T cell receptor gene previously described primers 5’-GTA
GGC ACC TGT GGG GAA GAA ACT-3’ and 5’-TGA GAG CTG TCT CCT ACT ATC
GAT T-3’ were used. Amplification was performed with 30 cycles (1 min at 95°C, 1
min at 69°C, and 1.5 min at 72°C) of PCR. PCR products were visualized by
electrophoresis in 1.8 % agarose gels with ethidium bromide. Agarose gels with
sample DNA were subsequently subjected to Southern blotting. Transferred
membranes were hybridized overnight at 60°C in 6X SSC , 5X Denhardt's solution,
0.5 %SDS, and 100µg/ml denatured salmon sperm DNA (Gibco BRL) using a
combination of 2
32
P- labeled DNA probes (40 p moles total) specific for Dβ- Jβ
gene segments. Probe 1: 5’-TTT CCC TCC CGG AGA TTC CCT AA-3’ and Probe
2: 5’-CGT CTT TAC CCT GAG TTC CCA AG-3’ were used (Anderson 1992).

FACS analysis.
Thymus, and spleen were obtained, and single cell suspensions were
prepared by teasing thymuses by using 26 gauge needles in PBS or passing
spleens through 70 µm nylon membrane cell strainers (Becton Dickinson Labware,
Franklin Lakes, NJ). Bone marrow cells were obtained by flushing femurs with PBS.

24
Cells were stained using mAbs for 30 min on ice and washed with 2 ml 1X PBS.
The following monoclonal antibodies were used for staining: FITC-, PE-, APC-, or
PerCP-conjugated anti-CD3ε, CD4, CD8, CD19, CD25, CD44, CD45.2, CD117, DX-
5, Gr-1, IgM, Mac-1, Ter119, and Thy1.2. All mAbs were obtained from Pharmingen
(San Diego, CA). Stained cells were analyzed using a FACS Calibur flow cytometer
(Becton Dickinson, Mountain View, CA) and CellQuest software (Becton Dickinson).
For analysis of earliest T-lineage progenitors (ETPs), FITC-, PE-, PE-Cy5-, PE-Cy7-,
or APC-conjugated anti-CD3ε, CD4, CD8α, CD8β, CD25, CD44, B220, Nk1.1, Mac-
1, TCRβ, TCRγ, IL-7Rα, c-kit, and Sca-1 were used for staining. Cells were
analyzed using a FACS Diva flow cytometer (Becton Dickinson, Mountain View,
CA) and Flowjo software (Tree Star, Inc. Ashland, OR).

Histological analysis.
Thymuses and spleens were fixed in 10% buffered formaldehyde solution
(Richard-Allan Scientific, Kalamazoo, MI) for 24 hours. Tissue sections were
prepared as described previously (Cao 1995) and were stained using hematoxylin-
eosin. For cytokeratin expression analyses, frozen thymus sections were stained
with rabbit anti-mouse keratin 5 (Covance Research Products Inc., Berkeley, CA)
and rat anti-keratin 8 (Troma-1, Developmental Studies Hybridoma Bank, University
of Iowa, Iowa City, IA) antibodies. FITC-donkey anti-rat IgG and Cy5-donkey anti-
rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) were
used as secondary antibodies. Immunofluorescence images were acquired using a
Leica DM microscope (Leica Microsystems, Wetzlar, Germany).

25
Statistical analysis.
Statistical analyses of data were performed by two-tailed t- test with unequal
distributions using Microsoft Excel software (Microsoft, Redmond, WA).

2.4 Results

Breeding of IL-7
-/-
Kit
W41/W41
and IL-7Rα
-/-
Kit
W41/W41
mice.
The IL-7
-/-
Kit
W41/W41
mice were generated by crossing IL-7 heterozygous IL-
7
wt/-
Kit
wt/W41
mice. The doubly mutant mice had the typical homozygous pigmentary
defect (white coat) of the Kit
W41/W41
strain. To further confirm that the mice had the
Kit
W41/W41
mutation, RT-PCR, PCR, and Xcm I restriction enzyme digestion were
performed as described in materials and methods (Figure 2.1). PCR of tail-vein
DNA for the IL-7 and IL-7Rα targeting vectors established which of the Kit
W41/W41

mice were also IL-7
-/-
or IL-7Rα
-/-
(Figure 2.1). The IL-7
-/-
Kit
W41/W41
and IL-7Rα
-/-
Kit
W41/W41
mice were viable. The mean weights for the adult IL-7
-/-
Kit
W41/W41
and IL-
7Rα
-/-
Kit
W41/W41
mice were 25.0 and 27.6 grams, respectively, which is 125% and
138% of the weight of the wild type mice, respectively. The fertility of the mice was
abnormal, with only 77% of breeding pairs producing at least one litter. However,
the fertility of the IL-7
-/-
Kit
W41/W41
and IL-7Rα
-/-
Kit
W41/W41
mice did not differ
significantly from that of the Kit
W41/W41
parental strain (data not shown).

26
Thymic hypoplasia in IL-7
-/-
Kit
W41/W41
and IL-7Rα
-/-
Kit
W41/W41
mice.
The mean thymic cellularity of the IL-7
-/-
Kit
W41/W41
and IL-7Rα
-/-
Kit
W41/W41
mice
was equivalent and significantly lower than that of either the normal mice or the
parental strains (Figure 2.2). The Kit
W41/W41
mice had an approximately 50%
reduction in thymic cellularity, which is similar to that reported for the Kit
W/W
strain
previously. The IL-7
-/-
mice had approximately 2-3% and the IL-7Rα
-/-
mice 1% of
the normal thymic cellularity. Consistent with previous observations, the thymic
cellularity of the IL-7Rα
-/-
mice is less than that of the IL-7
-/-
mice. In contrast, the
IL-7
-/-
Kit
W41/W41
and IL-7Rα
-/-
Kit
W41/W41
mice each had fewer than 300,000 total thymic
cells. Significantly, most of the thymic cells from both of the doubly mutated mice
were stromal elements. Both of the doubly mutated mice had fewer than 200 total
Thy1+ thymocytes, which is a 5-6 log reduction from normal. The IL-7
-/-
Kit
W41/W41

and IL-7Rα
-/-
Kit
W41/W41
mice were examined at age ranges from neonate to >20
weeks. No statistically significant difference in thymic cellularity was observed in the
time period (data not shown).

27

Kit
W41/W41

IL-7Rα
IL-7
KO
WT
KO
WT
WT
w41
wt
Kit
W41/W4
1

IL-7
-/-

IL-7Rα
-
/-

IL-7
-/-
Kit
W41/W4
1

IL-7Rα
-/-
Kit
W41/W4
1

Figure 2.1. PCR genotyping of the mouse strains used for this study.
PCR genotyping results using tail vein DNA and specific primers confirmed
that all the strains have the expected genotypes.
H
2
0

28
Figure 2.2 Synergistic effects of IL-7Rα
-/-
and Kit
W41/W41
mutations in
thymopoiesis. Effects of IL-7Rα
-/-
and Kit
W41/W41
mutations in thymopoiesis was
observed by differences in total thymocytes number of the six strains of mice. To
examine the effect of combined IL-7
-/-
or IL-7Rα
-/-
and Kit
W41/W41
mutations in
thymocyte number, IL-7
-/-
Kit
W41/W41
and IL-7Rα
-/-
Kit
W41/W41
mice were generated.
Numbers of mice per group are indicated in the parentheses. Thymuses were
obtained from mice and the number of thymocytes was counted by using a
hemocytometer. The data are expressed as the mean thymocyte number and +1
SD.

120
100
80
60
40
20
0
96.6M
55.5M
570K 380K 150 130

IL-7
IL-7R
Kit
(n)
wt
wt
wt
(5)
wt
wt
W41
(6)
KO
wt
wt
(5)
wt
KO
wt
(5)
KO
wt
W41
(6)
wt
KO
W41
(8)
Thy1 (CD90)+ cells
(X10
-6
)

29
Blocked prothymocyte differentiation in IL-7
-/-
Kit
W41/W41
and IL-7Rα
-/-
Kit
W41/W41
mice.
Thymocytes of the IL-7
-/-
Kit
W41/W41
and IL-7Rα
-/-
Kit
W41/W41
mice were examined to
determine whether they had reduced numbers of all thymic subsets or a block at a
specific stage of differentiation. The Kit
W41/W41
mice have no detectable alterations
in proportions of TN, DP or SP cells (Figure 2.3). The IL-7
-/-
and IL-7Rα
-/-
mice had
evidence of altered maturation with increased percentage of TN cells and lower
percentages of DP cells (Figure 2.3). In contrast, the IL-7
-/-
Kit
W41/W41
and IL-7Rα
-/-
Kit
W41/W41
mice had a complete absence of DP thymocytes. Both of the doubly
mutated mice had a small subpopulation of CD8+ SP cells in the thymus, which did
not express CD3 or the natural killer cell antigens NK-1.1 or DX5 (data not shown).
The block in pro-thymocyte differentiation in the IL-7
-/-
Kit
W41/W41
and IL-7Rα
-/
Kit
W41/W41

mice was at DN1, as determined by CD44 and CD25 expression (Figure 2.4). The
parental IL-7
-/-
mice have cells at both DN1 and DN2 while the IL-7Rα
-/-
mice have
pro-thymocytes predominantly at DN1. In contrast, both of the doubly mutated mice
both have only cells at Stage I of pro-thymocyte differentiation. Thus, loss of
function of the c-kit signaling pathway causes pro-thymocyte development in the IL-
7
-/-
Kit
W41/W41
mice to more closely resemble that of the IL-7Rα
-/-
and IL-7Rα
-/-
Kit
W41/W41
mice than that of the parental IL-7
-/-
mice (Figure 2.4).
Lack of repertoire formation in IL-7
-/-
Kit
W41/W41
and IL-7Rα
-/-
Kit
W41/W41
mice.
TCRβ gene rearrangements occur at the pro-thymocyte Stage II of
differentiation. Using the TCR Dβ2-Jβ2 family of rearrangements as a representative,
TCRβ rearrangements in the IL-7
-/-
Kit
W41/W41
and IL-7Rα
-/-
Kit
W41/W41
mice were

30

Figure 2.3. Blocked thymocyte differentiation in IL-7
-/-
Kit
W41/W41
and
IL-7Rα
-/-
Kit
W41/W41
mice. Thymocytes from the six strains of mice were stained
with PE-conjugated anti-CD8 and FITC-conjugated anti-CD4 antibodies
and analyzed using a FACS Calibur. Both doubly mutated mice have no
CD4+CD8+ double positive thymocytes.

Normal
IL-
7
-/-

IL-7
-/-

IL-7
-/-
Kit
W41/W41
CD4
IL-7Rα
-/-

Kit
W41/W41
IL-7Rα
-/-
Kit
W41/W41

2 91
6.5
2.2 91.8
5.4
IL
7
-/-
IL-
6.3 73.7
13.5
-
3.1 80.5
16
39.4
0
2.6 57.9
W41/W41
40.7
59.3
0
0
CD8

31

Figure 2.4 Differentiation block in DN thymocytes of IL-7-/- Kit
W41/W41
and IL-7Rα-/- Kit
W41/W41
mice. CD4 -CD8- double negative Thy-1
positive thymocytes (wild type, Kit
W41/W41
, IL-7-/- and IL-7Rα-/-)
or total Thy-1 positive thymocytes IL-7-/- Kit
W41/W41
and IL-7Rα-/- Kit
W41/W41
were stained with PE-conjugated anti-CD44 and FITC-conjugated
anti-CD25 antibodies. In IL-7-/- mice, thymocyte development is partially
blocked at the transition from DN 2 to DN 3. In the doubly mutated mice,
thymocyte development is blocked at the transition from DN1 to DN2.

CD25
Normal
IL-7
-/-

IL-7
-/-
Kit
W41/W41
Kit
W41/W41
IL-7Rα
-/-

IL-7Rα
-/-
Kit
W41/W41

CD44

32

Figure 2.5. Lack of TCR Dβ-Jβ gene rearrangement in IL-7
-/-
Kit
W41/W41
mice.
Lack of TCR Dβ-Jβ gene rearrangement in IL-7
-/-
Kit
W41/W41
mice. was shown
by the absence of Dβ2-Jβ2.1 to Dβ2-Jβ2.7 gene rearrangements. The
gene rearrangements were detected by a PCR analysis using primers
specific for these TCR segments.

IL-7
-/-
Kit
W41/W41

IL-7
-/-

Kit
W41/W41

H
2
O
O
normal
thymus
normal
liver
Germline
Dβ2-Jβ2.1
- - Dβ2-Jβ2.2
-
Dβ2-Jβ2.3
-
Dβ2-Jβ2.4
- Dβ2-Jβ2.5
-
Dβ2-Jβ2.7
-

33

Figure 2.6 Lack of TCR Dβ-Jβ gene rearrangement in IL-7Rα
-/-
Kit
W41/W41
mice. Lack of TCR Dβ-Jβ gene rearrangement in IL-7Rα
-/-
Kit
W41/W41
mice was
shown by the absence of Dβ2-Jβ2.1 to Dβ2-Jβ2.7 gene rearrangements.
The gene rearrangements were detected by a PCR analysis using primers
specific for these TCR segments.

IL-7Rα
-/-
Kit
W41/W41

IL-7Rα
-/-

Kit
W41/W41

H
2
O
normal
thymus
normal
liver
Germline
Dβ2-Jβ2.1
Dβ2-Jβ2.2
Dβ2-Jβ2.3
Dβ2-Jβ2.4
Dβ2-Jβ2.5
Dβ2-Jβ2.7

34
characterized (Figures 2.5 and 2.6). The thymuses of the IL-7
-/-
, Kit
W41/W41
and

normal mice contained a full range of TCR Dβ2-Jβ2 rearrangements. The IL-7Rα
-/-

mice had detectable rearrangements only of the TCR Dβ2-Jβ2.4, Dβ2-Jβ2.5, and
Dβ2-Jβ2.6 segments, consistent with previous observations of defects in V(D)J
rearrangements in the IL-7Rα
-/-
mice. Unlike the IL-7
-/-
and Kit
W41/W41
parental
strains, the IL-7
-/-
Kit
W41/W41
mice had a complete absence of TCR Dβ2-Jβ2 gene
rearrangements (Figure 2.5). The IL-7Rα
-/-
Kit
W41/W41
mice also had a complete loss
of TCR Dβ2-Jβ2 rearrangements (Figure 2.6).

Reduced early T-lineage progenitor frequency in IL-7Rα
-/-
, IL-7
-/-
Kit
W41/W41
, and
IL-7Rα
-/-
Kit
W41/W41
mice.
In order to examine the nature of DN1 pro-thymocytes in mutant mice further,
frequency of early T-lineage progenitors (ETPs) in mutant mice and control strains
was determined. ETPs are defined as CD44
hi
lin
-
CD25
-
IL-7Rα
lo/-
c-kit
hi
Sca-1
hi
and
possessing a high potential to produce T-lineage cells. There was no significant
difference between ETP frequency of wild type, Kit
W41/W41
, or IL-7
-/-
mice. Compared
to these strains, ETP frequency of IL-7Rα
-/-
,

IL-7
-/-
Kit
W41/W41
, and IL-7Rα
-/-
Kit
W41/W41

mice was significantly reduced (P<0.05). Introduction of the bcl-2 transgene did not
increase ETP frequency of Kit
W41/W41
, IL-7
-/-
, IL-7Rα
-/-
, IL-7
-/-
Kit
W41/W41
, or IL-7Rα
-/-
Kit
W41/W41
mice (P>0.05) (Figure 2.7).

35

Figure 2.7 Reduced early T-lineage progenitor frequency in IL-7Rα
-/-
, IL-7
-/-
Kit
W41/W41
, and IL-7Rα
-/-
Kit
W41/W41
mice. Thymocytes from the indicated mouse
strains were analyzed for frequency of ETP, defined as percent of c-kithi Sca-1hi IL-
7R-/lo cells within CD44hi lin- cell population. Asterisks indicate that thymuses were
pooled to obtain sufficient number of thymocytes to be analyzed.

IL-7
IL-7R
Kit
Tg bcl-2
(n)
wt
wt
wt
-
(7)
wt
wt
W41
-
(7)
KO
wt
wt
-
(6)*
wt
KO
wt
-
(6)*
wt
KO
wt
+
(7)
wt
KO
W41
-
(6)*
wt
KO
W41
+
(3)*
wt
wt
W41
+
(3)
KO
wt
wt
+
(3)
KO
wt
W41
-
(6)
KO
wt
W41
+
(3)*
ETP frequency
(%)

36
2.5 Discussion
We have generated IL-7
-/-
Kit
W41/W41
and IL-7Rα
-/-
Kit
W41/W41
mice to test the
hypothesis that c-kit and IL-7R signaling have synergistic roles in thymopoiesis. The
doubly mutated mice have a significant loss of thymic cellularity with evidence of a
complete block in pro-thymocyte differentiation compared to the parental strains.
The thymic cellularity and differentiation block are equal in both of the doubly
mutated mice. Thus, the IL-7R and c-kit signaling pathways are synergistic and
partially redundant for the differentiation, proliferation and/or survival of immature
thymocytes. The reduction in Thy-1 positive thymocyte numbers, block in pro-
thymocyte differentiation at stage I, and abrogation of thymic phenotypic differences
betweenIL-7
-/-
and IL-7Rα
-/-
mice when the Kit
W41/W41
mutation is introduced into
these parental strains all suggest that IL-7/IL-7R and KL/Kit pathways have
synergistic and partially redundant functions in thymopoiesis.
Although Kit
W41/W41
mice have 50% of normal thymus cellularity, IL-7
-/-
mice
have 2-3%, and IL-7Rα
-/-
mice have 0.5%, respectively, both strains of doubly
mutated mice have a 5-6 log reduction in thymus cellularity compared to normal.
The decreased thymopoiesis in the doubly mutated mice compared to Kit
W41/W41
, IL-
7
-/-
, and IL-7Rα
-/-
parental strains indicates that the c-kit and IL-7R may be partially
redundant. Similar thymic failure as seen in the doubly mutated mice was also
demonstrated in γc
-
Kit
-/-
mice by Rodewald et al (1997). Since the γc subunit of the
IL-7R was deleted in the mice, it was not possible to determine which of the γc-
containing cytokine receptors is responsible for the thymic phenotypes observed.
Because the Kit
-/-
mutation is neonatally lethal, it was also not possible to determine

37
whether there was leakiness of the thymopoietic defect in the γc
-
Kit
-/-
mice
(Rodewald 1997). In the IL-7
-/-
Kit
W41/W41
and IL-7Rα
-/-
Kit
W41/W41
mice, the
thymopoietic defect was equally severe in all ages from birth to greater than 16
weeks. Thus, the introduction of the c-kit mutation into IL-7
-/-
or IL-7Rα
-/-
background
eliminates the leakiness of the thymic defect in the IL-7
-/-
or IL-7Rα
-/-
parental strains
(von Freeden-Jeffry 1995, Peschon 1994).
IL-7
-/-
mice have less severe thymic defects than IL-7Rα
-/-
mice indicating
that there is a possibility of the presence of an alternative signaling pathway
involved in thymopoiesis. One possible pathway is the thymic stroma-derived
lymphopoietin (TSLP). TSLP activates a receptor complex consisting of the IL-7Rα

and a TSLP-specific receptor subunit (von Freeden-Jeffry 1995, Park 2000). TSLP
was originally cloned from thymic stromal cells of IL-7
-/-
mice, but has also been
found in other stromal sources (Friend 1994). The TSLPR signaling might provide
an alternative pathway for thymic differentiation that would not be abrogated in IL-7
-/-

mice but would be in IL-7Rα
-/-
mice. TSLP has effects on both B and T cells, which
are still being elucidated (Levin 1999). The phenotypic differences between IL-7
-/-

and IL-7Rα
-/-
mice might be due to the ability of TSLP to complement the thymic
defects in the absence of IL-7. In this model, TSLP signaling through the IL-7Rα
and TSLPR would be intact in IL-7
-/-
mice but disrupted in IL-7Rα
-/-
mice. However,
our results show that TSLP is probably not critical for pro-thymocyte expansion and
differentiation because both IL-7
-/-
Kit
W41/W41
and IL-7Rα
-/-
Kit
W41/W41
mice which should
have an intact or defective TSLP pathway, respectively, have an identical block in
pro-thymocyte differentiation (at DN1) and expansion. If TSLP was
critical for pro-thymocyte differentiation, the two strains of doubly mutated mice

38
should have different thymic phenotypes. Alternatively, it is possible that TSLP
might be involved in later stage(s) of thymic differentiation.
The data from this study suggest the possibility that IL-7/IL-7R and KL/c-kit
pathways overlap. Other studies from the Weinberg laboratory (Jahn 2007) show
that there is physical interaction between the two pathways. Recently interaction
between IL-7R and c-kit and activation of IL-7R in the absence of IL-7 ligation by
activated c-kit in vitro has been identified (Jahn 2007). We propose that this
interaction between IL-7R and c-kit in mouse thymocytes may account for the
phenotypic differences in the thymuses of IL-7
-/-
and IL-7Rα
-/-
mice. The difference
in the thymic phenotypes of IL-7
-/-
and IL-7Rα
-/-
mice could be due to the presence
or absence of complementary association, respectively, between IL-7R and c-kit. In
IL-7Rα
-/-
mice, which have more severe thymic defects than IL-7
-/-
mice, there is no
possibility of association between IL-7R and c-kit whereas in IL-7
-/-
mice, the
interaction might still occur in the absence of IL-7.
An example of similar interactions between a receptor tyrosine kinase and a
cytokine receptor is the direct interaction of c-kit and two other type I cytokine
receptors, the erythropoietin receptor (EpoR) and IL-3R (Wu 1995, Linnekin 1995).
Wu and others showed that KL induced tyrosine phosphorylation of the EpoR in
HCD57 cells which are an Epo-dependent erythroid progenitor cell line cells with a
high level of Kit expression (Wu 1995). This experiment showed that KL can
replace Epo in supporting the growth and survival of HCD57 cells through activation
of EpoR by tyrosine phosphorylation (Wu 1995). In these experiments, activation of
the EpoR occurred after c-kit stimulation, even in the absence of Epo. This showed
that a physical association of c-Kit with the cytoplasmic domain of the EpoR exists

39
(Wu 1995). C-kit activates multiple signal transduction pathways including PI3K,
Src family members, the Jak/STAT pathway and the Ras-Raf-MAPK cascade
(Linnekin 1999). In addition to JAK kinases, which are responsible for
phosphorylation of STATs, Src family kinases are involved in phosphorylation of
STATs following IL-3 binding to its receptor (Reddy 2000).

In epidermal growth
factor (EGF) signaling, STAT activation is dependent on Src but not JAK kinases
(Olayioye 1999). STAT proteins function as substrates for MAP kinases, and this
phosphorylation can enhance the transcriptional activity of STATs (Rane 2002).
These data indicate that Src kinases have important functions in cytokine receptor-
and receptor tyrosine kinase-mediated signaling including c-kit. Involvement of Src
family kinases in both IL-7R and c-kit signaling pathways suggest that Src may
function to connect the pathways and may be partially responsible for the synergistic
action of the two pathways in thymopoiesis. In addition, experimental data from the
Weinberg laboratory indicate that IL-7R and c-kit are co-localized in lipid rafts after
KL stimulation in vitro (Jahn 2007). Physical interaction between IL-7R and c–kit
and activation of IL-7R in the absence of IL-7 by activated c-kit may explain the
synergism between IL-7R and c-kit pathways which is observed in thymopoiesis.
The interaction between IL-7R and c-kit may be responsible for the proposed
compensatory mechanism in cytokine (IL-7) deficient mice but not in cytokine
receptor (IL-7Rα) deficient mice because in IL-7Rα
-/-
mice, ligand-independent
activation of IL-7R mediated by c-kit cannot occur.

Early T-lineage progenitors, ETP, are recently identified putative T lineage
progenitors in the thymus (Allman 2003). In order to elucidate functional roles of IL-

40
7R and c-kit signaling pathways at this very early stage of thymopoiesis, frequency
and absolute numbers of ETP in different stains of mice analyzed in this study were
examined. There was no significant difference between ETP frequency of wild
type, Kit
W41/W41
, or IL-7
-/-
mice. Compared to these strains, ETP frequency of IL-7Rα
/-
,

IL-7
-/-
Kit
W41/W41
, and IL-7Rα
-/-
Kit
W41/W41
was significantly reduced (P<0.05).
Introduction of the bcl-2 transgene did not increase ETP frequency of Kit
W41/W41
, IL-
7
-/-
, IL-7Rα
-/-
, IL-7
-/-
Kit
W41/W41
, or IL-7Rα
-/-
Kit
W41/W41
mice indicating that survival,
proliferation, and/or differentiation of ETP is not affected by enforced expression of
bcl-2. A recent study has shown that LSK (lin- Sca-1+ c-kit+) progenitors may not be
precursors of ETP (Benz 2008). Because there is presently no consensus in the
origin and nature of T-lineage progenitors within the thymus, further studies in this
area are necessary.

41
Chapter 3

Determination of nature of IL-7R/c-kit signaling pathways - permissive or
instructive action – through transgenic expression of Bcl-2 in thymocytes

3.1 Abstract
Results of the chapter 2 of this study demonstrate that IL-7R and c-kit
signaling in thymopoiesis have synergistic, partially redundant and unique functions.
Over-expression of anti-apoptotic bcl-2 in growth factor-dependent cell lines was
used to determine the nature of signaling pathways. In order to determine the nature
of these signaling pathways – either permissive or instructive – in thymopoiesis,
transgenic bcl-2 was expressed in thymocytes of different genotypes used in this
study. Introduction of the bcl-2 transgene in IL-7Rα
−/−
or IL-7Rα
−/−
Kit
W41/W41
mice
resulted in 16.4-fold (4.2x10
5
vs. 6.9x10
6
) or 80-fold (250 vs. 2x10
4
) increase in Thy-
1 positive thymocyte number, respectively. The block in differentiation of DN1
thymocytes in IL-7Rα
−/−
Kit
W41/W41
mice was not significantly relieved by the
introduction of the bcl-2 transgene. However, in IL-7Rα
−/−
Kit
W41/W41
mice, the
transgene relieved the block in differentiation between DN and DP stages, and DN
thymocyte numbers of IL-7Rα
−/−
Kit
W41/W41
mice was increased 8-fold. Collectively,
these data suggest that in order for the bcl-2 transgene to exert its effect, the
presence of a functional c-kit is necessary. This suggests that c-kit provides
instructive signals in thymopoiesis.

42
3.2 Introduction

Studies on IL-7-/-, IL-7Rα-/-, and Kit mutant mice have demonstrated that IL-
7R and c-kit signaling is required for thymopoiesis (von Freeden-Jeffry 1995,
Peschon 1994, Rodewald 1995). A study by Rodewald (1997) has shown that the
cytokinre receptor common gamma chain (γc) and c-kit signaling are essential for
thymopoiesis. However, the nature and roles of signaling pathways downstream of
γc and c-kit have only been partilally elucidated.
Over-expression of the antiapoptotic regulator B-cell lymphoma-2 (BCL2) in
growth factor dependent hematopoietic cell lines can result in their independence
from hematopoietin family cytokines for survival (Fairbairn 1993). In vivo studies
demonstrated that defective thymopoiesis in interleukin-7 (IL-7) and IL-7Rα–
deficient (IL7-/- and IL7Rα-/-) mice can be rescued by transgenic expression of
BCL2 (Akashi 1997, Kondo 1997, Maraskovsky 1997). This supports the hypothesis
that hematopoietin family cytokine receptors such as IL-7R play a permissive role in
thymopoiesis. Hematopoiesis is also regulated by a distinct family of cytokines
whose signals are mediated through tyrosine kinase receptors, such as c-kit and
FMS-like tyrosine kinase 3 (Flt3) (Lyman 1998). Although c-kit, similar to IL-7Rα, is
critically involved in the regulation of thymopoiesis, BCL2 over-expression failed to
rescue the impaired T lymphopoiesis in c-kit-deficient mice (Rodewald 2001). This
implies that signaling mediated by some cytokine receptors and ligands, including c-
kit and stem cell factor, unlike the hematopoietin family, might not promote
hematopoiesis through permissive actions (Jensen 2008).

43
Survival of thymocytes in vivo is dependent on the expression of anti-
apoptotic proteins such as bcl-2, and its expression is induced downstream of IL-7R
signaling (von Freeden-Jeffry 1997) through STAT5 at all stages of cells
undergoing positive selection, including DN and SP cells, but not in the DP cells that
have failed positive selection and that will die by default. Bcl-2 deficient mice
showed a gradual decline in T and B cells after birth and a severe progressive
thymic atrophy due to massive apoptosis, suggesting that bcl-2 may protect
thymocytes undergoing positive selection as well as peripheral T cells from
apoptotic stimuli such as glucocorticoids. The enforced expression of bcl-2 has been
shown to maintain the viability of various cytokine-dependent cells upon cytokine
withdrawal in vitro. In these systems, bcl-2 does not stimulate proliferation, but it
appears to enable cells to undergo intrinsically-determined differentiation through
maintenance of the viability of the cells in vitro (Akashi 1997).
Biochemical and genetic experiments have shown that the IL-7R and c-kit
directly interact (Jahn 2007), suggesting that the intact c-kit pathway may
complement the loss of IL-7R signaling in IL-7Rα-/- transgenic bcl-2 mice. In the
absence of functional c–kit pathway, a bcl-2 transgene does not exert its protective
effects in terms of thymocyte cellularity and differentiation (Rodewald 2001). Based
on these observations, we hypothesized that the thymopoietic defect of IL-7Rα
-/-
Kit
W41/W41
mice cannot be complemented by transgenic bcl-2 expression. The studies
presented in this chapter evaluated whether enforced expression of an anti-
apoptotic gene, bcl-2, could maintain thymopoiesis in the absence of IL-7R and/or c-
kit signaling which have been shown to be essential for thymopoiesis.

44
3.3 Materials and Methods
Breeding of IL-7
-/-
Kit
W41/W41
tg bcl-2 and IL-7Rα
-/-
Kit
W41/W41
tg bcl-2 mice.
C57BL/6-Tg(BCL2)36Wehi/J (Eµ-bcl-2-36) transgenic mice (Jackson
Laboratory, Bar Harbor, ME) on a C57BL/6J background in which the human bcl-2
cDNA is expressed by Eµ immunoglobulin heavy chain gene enhancer and SV40
promoter were bred with Kit
W41/W41
, IL-7
-/-
, IL-7
-/-
Kit
W41/W41
, IL-7Rα
-/-
or IL-7Rα
-/-
Kit
W41/W41
mice to generate IL-7
-/-
, IL-7
-/-
Kit
W41/W41
,IL-7Rα
-/-
or IL-7Rα
-/-
Kit
W41/W41
transgenic bcl-2 mice. Genotyping of mice for expression of the transgene was
performed by PCR using primers A: 5’-CTA GGC CAC AGA ATT GAA AGA TCT-3’,
B: 5’-GTA GGT GGA AAT TCT AGC ATC ATC C-3’, C: 5’-TGG ATC CAG GAT
AAC GGA AA-3’, and D: 5’-TGT TGA CTT CAC TTG TGG CC-3’ (Jackson
Laboratory, Bar Harbor, ME). Primers A and B produce a 315bp band which is used
as a loading control, and primers C and D produce a 170bp band if a mouse
possesses the bcl-2 transgene.

FACS analysis.
Thymuses of the bcl-2 transgenic strains as well as control wildtype mice at
4 to 8 weeks of age were obtained, and single cell suspensions were prepared by
teasing thymuses by using 26 gauge needles in PBS or passing spleens through 70
µm nylon membrane cell strainers (Becton Dickinson Labware, Franklin Lakes, NJ).
Bone marrow cells were obtained by flushing femurs with PBS. Cells were stained
using mAbs for 30 min on ice and washed with 2 ml 1X PBS. The following
monoclonal antibodies were used for staining: FITC-, PE-, APC-, or PerCP-
conjugated anti-CD3ε, CD4, CD8, CD19, CD25, CD44, CD45.2, CD117, DX-5, Gr-1,

45
IgM, Mac-1, Ter119, and Thy1.2. All mAbs were obtained from Pharmingen (San
Diego, CA). Stained cells were analyzed using a FACS Calibur flow cytometer
(Becton Dickinson, Mountain View, CA) and CellQuest software (Becton Dickinson).
For analysis of earliest T-lineage progenitors (ETPs), FITC-, PE-, PE-Cy5-, PE-Cy7-,
or APC-conjugated anti-CD3ε, CD4, CD8α, CD8β, CD25, CD44, B220, Nk1.1, Mac-
1, TCRβ, TCRγ, IL-7Rα, c-kit, and Sca-1 were used for staining. Cells were
analyzed using a FACS Diva flow cytometer (Becton Dickinson, Mountain View,
CA) and analyzed with Flowjo software (Tree Star, Inc. Ashland, OR).

3.4 Results
Effect of transgenic bcl-2 expression in thymocyte proliferation and differentiation.
It has been shown that in the absence of a functional c-kit signaling,
transgenic expression of anti-apoptotic bcl-2 cannot rescue thymopoiesis (Rodewald
2001). To test the hypothesis that the thymopoietic defect of IL-7Rα
-/-
Kit
W41/W41
mice
cannot be complemented by transgenic bcl-2 expression, IL-7Rα
-/-
transgenic bcl-2
mice and IL-7Rα
-/-
Kit
W41/W41
transgenic bcl-2

mice were generated and thymocyte
numbers and differentiation of the mice were examined.
Introduction of a bcl-2 transgene (Eµ-bcl-2-36) in which the human bcl-2
cDNA is expressed by Eµ immunoglobulin heavy chain gene enhancer and SV40
promoter and is over-expressed in both T and B lineages into IL-7Rα
−/−
or IL-
7Rα
−/−
Kit
W41/W41
mice resulted in 16.4-fold (4.2x10
5
vs. 6.9x10
6
) or 80-fold (250 vs.
2x10
4
) increase in Thy-1 positive thymocyte number, respectively. These thymocyte
numbers are significantly smaller than that of wild type mice (114x10
6
). (Figure 3.1)
The block in differentiation of DN thymocytes observed in IL-7Rα
−/−
mice was

46
modestly reduced by expression of the bcl2 transgene. In IL-7Rα
−/−
mice, the DN
thymocytes were predominantly in the DN1 stage (54.2% DN1, 27.4% DN2, 0%
DN3, and 18.4% DN4; total average DN thymocyte number: 2094), while in the IL-
7Rα
−/−
transgenic bcl2 mice, an increase in DN2 thymocytes was observed (45.0%
DN1, 37.7% DN2, 2.9% DN3, 14.2% DN4; total average thymocyte number:
4.0x10
5
). (Figure 3.2)
However, the DN1 to DN2 block in differentiation found in IL-7Rα
−/−
Kit
W41/W41
mice was not significantly reduced by the transgene (IL-7Rα
−/−
Kit
W41/W41
mice, 90.0%
DN1, 2.4% DN2, 0% DN3, 7.6% DN4; total average thymocyte number: 250; IL-
7Rα
−/−
Kit
W41/W41
tg bcl-2 mice, 65.9% DN1, 19.6% DN2, 1.3% DN3, 13.1% DN4; total
average thymocyte number: 2x10
4
). (Figure 3.2) In terms of DN to DP
differentiation, no significant change was observed in IL-7Rα
−/−
mice as a result of
introduction of the bcl-2 transgene into the IL-7Rα
−/−
background (IL-7Rα
−/−
mice,
14.8% DN, 56.3% DP; IL-7Rα
−/−
tg bcl-2 mice, 0.8% DN, 79.5%% DP). However, in
IL-7Rα
−/−
Kit
W41/W41
mice, the transgene relieved the block in differentiation between
DN and DP stages (IL-7Rα
−/−
Kit
W41/W41
mice, 51% DN, 2% DP; IL-7Rα
−/−
Kit
W41/W41
tg
bcl-2 mice, 0.3% DN, 63.8% DP). (Figure 3.3) DN thymocyte numbers of IL-
7Rα
−/−
Kit
W41/W41
mice, 66, was increased to 526, an 8-fold increase.

47

Figure 3.1 Thymocyte numbers were significantly increased by the bcl-2
transgene in the presence of a functional c-kit. Thymocytes from the indicated
mouse strains were analyzed for the number of thymocytes defined as CD90 (Thy-
1) positive cells.

25
0
20K
114M
63M
420K 6.9M
120
100
80
60
40
20
0

140
IL-7R
Kit
Tg Bcl-2
(n)
wt
wt
-
(12)
wt
W41
-
(12)
KO
wt
-
(12)
KO
wt
+
(5)
KO
W41
-
(12)
KO
W41
+
(12)
Thymocytes (x10
-6
)

48

Figure 3.2 DN thymocyte differentiation block is not relieved by enforced
expression of bcl-2 when a functional c-kit is absent. DN thymocytes from the
indicated mice were examined for their expression of CD44 and CD25 and relative
frequency of each subset was determined.

49

Figure 3.3 Relieve of DN to DP differentiation block in IL-7Rα
−/−
Kit
W41/W41
mice
by the enforced expression of bcl-2. Total thymocytes from the indicated mice
were examined for their expression of CD4 and CD8 and relative frequency of
indicated subset was determined by flow cytometric analyses.

50
Discussion

Our results indicate that a functional c-kit is required for the bcl-2 transgene
to exert its effect on thymopoiesis - rescue hypocellularity and a block in
differentiation in IL-7Rα
−/−
Kit
W41/W41
tg bcl-2 mice. This is consistent with results of
Rodewald (2001) and Jensen (2008), supporting the idea that unlike the
hematopoietin family, some cytokine receptors and ligands including c-kit and SCF,
might promote hematopoiesis through instructive actions. Previous studies of IL-
7Rα-/-, or γc- transgenic bcl-2 mice (Akashi 1997, Kondo 1997) both of which have
an intact c-kit/SCF signaling pathway show that thymus cellularity and block in
differentiation of thymocytes can be rescued by the introduction of a bcl-2
transgene. Our analysis of IL-7
−/−
Kit
W41/W41
and IL-7Rα
−/−
Kit
W41/W41
thymocytes
indicates that the bcl-2 transgene does not rescue thymopoiesis when the Kit
W41/W41
mutation is introduced into either IL-7-/- or IL-7Rα-/- background. This suggests that
c-kit signaling pathway provides more than survival signals in thymopoiesis and
therefore c-kit may provide instructive signals.
This conclusion is not in accordance with a recent study which examined the
effect of introducing a bcl-2 transgene into Flt3l-/-Il7r-/- mice (Jensen 2008). In the
experiments presented in this Chapter, overexpression of BCL2 partially rescues all
stages of T-cell development. Only small increase in thymus cellularity and a
modest release in DN thymocyte differentiation block were observed in IL-
7Rα
−/−
Kit
W41/W41
tg bcl-2 mice compared to non-transgenic mice. This might be due to
the differences in Flt3- and c-kit-mediated signaling in thymocytes (Lyman 1998).

51
In addition, Jensen and others (2008) have shown that Flt3 signaling plays a
significant role in murine thymopoiesis only when IL-7R signaling is absent. This
suggests that the relative contribution of Flt3 to thymopiesis is smaller compared to
that of c-kit.
Other anti-apoptotic proteins such as Mcl-1 (Dzhagalov 2008) or Bcl-xL
(Jones 2000) have been shown to promote survival of DN thymocytes, and
activation and/or suppression of these proteins are mediated by ligation of cytokines
and growth factors to their cognate receptors and downstream signals of these
receptors.
The results of this current study has important implications in elucidation of
the nature of signaling pathways mediated by a hematopoietin receptor (IL-7R) and
a growth factor receptor (c-kit). Previous studies have suggested that IL-7R
signaling has permissive roles in murine thymopoiesis (Kondo 1997, Akashi 1997,
Maraskovsky 1997) whereas c-kit may have instructive roles (Rodewald 2001).
Proliferation and differentiation of IL-7Rα
−/−
Kit
W41/W41
DN thymocytes were minimally
increased by the introduction of a bcl-2 transgene (8-fold and Figure 3.1) even
though the same transgene significantly increased these parameters when
introduced into IL-7Rα-/- mice (193-fold and Figure 3.1). c-kit signaling is intact in
IL-7Rα-/- mice.
Regulation of proteins involved in apoptosis of thymocytes such as bcl-2,
Bcl-xL, and Bad is complex and developmentally controlled, and this regulation
involves multiple signaling pathways (Tomayko 1999). Interactions between the
signaling pathways involved in the regulation of pro- and anti-apoptotic proteins are
not well understood especially in DN thymocytes.

52
In an in vitro experiment using an OP9-DL1 culture system which allows
differentiation and proliferation of progenitor cells into the T lineage, after T lineage
specification by Notch, DN1 and DN2 prothymocytes require c-kit signaling, in
addition to Notch and IL-7R signaling, for proliferation and differentiation. Results of
the study presented in this chapter are consistent with these data, suggesting that
requirement of c–kit signaling in Notch and IL-7R-induced proliferation and
differentiation of DN1 and DN2 prothymocytes (Massa 2006) also exists in vivo.
In IL-7Rα
−/−
Kit
W41/W41
tg bcl-2 mice, DN to DP differentiation block was
relieved compared to IL-7Rα
−/−
Kit
W41/W41
mice. This may be due to the enhanced
survival and differentiation of DN thymocytes mediated by Flt3 signaling. Because
Flt3 signaling is intact in IL-7Rα
−/−
Kit
W41/W41
thymocytes, residual thymocytes are
expected to be present in IL-7Rα
−/−
Kit
W41/W41
mouse thymus (Jensen 2008). It is
possible that the small number of prothymocytes in IL-7Rα
−/−
Kit
W41/W41
thymus which
were generated by Flt3 signaling were allowed to survive by the enforced
expression of bcl-2. However, because DN thymocytes require IL-7R and c-kit
signaling for proliferation and differentiation, the bcl-2 transgene only exerted a
small increase (8-fold) in DN thymocyte numbers when introduced into IL-
7Rα
−/−
Kit
W41/W41
mice. In comparison, when introduced into IL-7Rα
−/−
mice, the same
bcl-2 transgene increased DN thymocyte number 193-fold. The phenotypic
differences between IL-7Rα
−/−
tg bcl-2 and IL-7Rα
−/−
Kit
W41/W41
tg bcl-2 mice indicate
that in order for the bcl-2 transgene to exert its effect and complement loss of
cytokine signaling required for thymopoiesis, the presence of a functional c-kit
signaling pathway is necessary. In addition, it is suggested that c-kit signaling

53
provides more than survival signals in thymopiesis, and c-kit may have instructive
roles in thymopoiesis.

54
Chapter 4

Effects of IL-7R and c-kit signaling on thymic architecture and thymic
epithelial cell differentiation

4.1 Abstract
The thymus is organized into medulla and cortex which have distinctive roles in
spedific stages of T-cell development. Thymic epithelial cells (TEC) have been
shown to play indispensable roles in thymopoiesis. Thymuses of mutant mice which
are alymphoid lack a three-dimensional cortical or medullary structure with absence
of cortico-medullary junction. In these hypoplastic thymuses, differentiation of TEC
which can be determined by expression pattern of cytokeratin (K) is blocked at the
immature K8+K5+ stage. K8 expression is specific to cortical TEC, and K5,
medullary TEC. It has been proposed that lymphostromal interactions between TEC
and developing thymocytes called thymic cross-talk is important for the development
of both thymocytes and TEC. Immunofluorescence analyses showed that the
differentiation of TEC in the thymuses of IL-7Rα
-/-
Kit
W41/W41
mice was arrested at the
immature K8+K5+ stage whereas thymuses of wild type mice and parental strains
had distinctive cortial and medullary areas. Enforced expression of bcl-2 in IL-7Rα
-/-
Kit
W41/W41
mice resulted in the emergence of K8+K5- medullary areas.

55
4.2 Introduction
The thymus is organized into specialized medullary and cortical zones which have
distinctive roles in spedific stages of T-cell development (Rodewald 2001).
Thymuses of γc
-
c-kit
w/w
mice which are alymphoid lack a three-dimensional cortical
or medullary structure with absence of cortico-medullary junction (Rodewald 1997).
The formation of three-dimensional medulla and cortex compartments is thought to
occur through interaction of an endodermal epithelium into an ectodermally derived
tissue (Rodewald 2001). Differentiation status of thymic epithelial cells (TEC) can be
determined by their expression pattern of cytokeratin (K) 8 or 5 (Klug 1998, Klug
2002). Immature TECs coexpress K8 and K5 (K8+K5+). K8 expression is specific to
cortical TEC, and K5, medullary TEC. TEC of mutant mice which have abnormal
thymopoiesis with massive early block in T cell development, such as c-kit
W/W
γc
-
,
human CD3ε transgenic, RAG2
−/−
γc
−
mice, expression pattern and distribution of K8
and K5 positive areas are abnormal and large clusters of K5+K8+ TEC are present
(Rodewald 2008). In these mutant mice, differentiation of most TEC was blocked at
the immature K8+K5+ stage, suggesting that the lack of developing thymocytes in
these thymuses may have caused the arrested development of thymic architecture
and TEC differentiation. Lymphostromal interactions between TEC and thymocytes,
known as thymic cross-talk, has important roles in proper development of thymic
architecture (Klug 1998). It has been demonstrated that down-regulation of K5 in the
thymic cortex depends on cross-talk between K8+K5+ TEC precursors and T
lineage-committed thymocytes (Klug 1998, Klug 2000). For example, although the
presence of hemopoietic cells is not required to initiate IL-7 expression during
thymic organogenesis, thymocyte-derived signals are necessary to maintain IL-7-

56
expressing TECs in the adult thymus (Zamisch 2005). Because IL-7 is required for
normal thymopoiesis, this study showed that thymic cross-talk is essential for proper
thymic function. In this chapter, thymic K8 and K5 expression pattern of different
mutant strains is examined in order to examine the effects of IL-7R and c-kit
signaling in thymic architecture and thymic epithelial cell differentiation.

4.3 Materials and Methods
Histological analysis.
Thymuses were fixed in 10% buffered formaldehyde solution (Richard-Allan
Scientific, Kalamazoo, MI) for 24 hours. Tissue sections were prepared as follows.
After mice were euthanized, thymuses were collected and placed in OCT medium
(Sakura Finetec, Torrance, CA). Tissue blocks were frozen by using liquid nitrogen
cooled methylbutane (Sigma) and were kept at -80°C. Frozen sections (7µm) were
made using a Leica 1900 cryostat (Leica Micosystems, Wetzlar, Germany) and
were stained using hematoxylin-eosin. For cytokeratin expression analyses, frozen
thymus sections were fixed with 4% paraformaldehyde solution (pH 7.4) for 5
minutes and air dried and washed in PBS. Then the slides were stained with rabbit
anti-mouse keratin 5 (Covance Research Products Inc., Berkeley, CA) and rat anti-
keratin 8 (Troma-1, Developmental Studies Hybridoma Bank, University of Iowa,
Iowa City, IA) antibodies. FITC-donkey anti-rat IgG and Cy5-donkey anti-rabbit IgG
(Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) were used as
secondary antibodies. Immunofluorescence images were acquired using a Leica
DM microscope (Leica Microsystems, Wetzlar, Germany).

57
4.4 Results
Histological analysis.
Gross anatomical examination showed that thymuses of IL-7
-/-
Kit
W41/W41
and
IL-7Rα
-/-
Kit
W41/W41
mice were visible but severely reduced in size. Histological
analyses of the thymuses of the parental strains (Figure 4.1) and doubly mutant
mice (Figure 4.1) showed that thymuses of the doubly mutated mice lacked a
distinctive corticomedullary junction and cortical and medullary regions compared to
normal thymus (Figure 4.1) or Kit
W41/W41
thymus (Figure 4.1). Cysts were observed
in the thymuses of IL-7
-/-
Kit
W41/W41
and IL-7Rα
-/-
Kit
W41/W41
mice. Thymuses of IL-7
-/-

Kit
W41/W41
and IL-7Rα
-/-
Kit
W41/W41
have similar histological characteristics.
Cytokeratin 5 and 8 expression pattern of the murine thymuses indicate that
thymic epithelial cells found in thymuses of wild type and parental strains consist of
cytokeratin 5 or 8 single positive as well as rare, immature double positive cells
localized in proximity to corticomedullary junction (Figure 4.2). In contrast, IL-7
-/-
Kit
W41/W41
and IL-7Rα
-/-
Kit
W41/W41
thymuses are consisted mostly of immature
cytokeratin 5 and 8 double positive thymic epithelial cells with absence of more
differentiated cytokeratin 5 or 8 single positive regions (Figure 4.2). Areas of
hypercellularity were observed in IL-7Rα
-/-
Kit
W41/W41
tg bcl-2 thymuses. These areas
are cytokeratin 5 single positive indicating that medullary regions developed in these
thymuses as a result of the introduction of the bcl-2 transgene and resultant
increase in thymic cellularity.

58

Wild type Kit
W41/W41

IL-7
-/-

IL-7Rα
-/-

IL-7
-/-
Kit
W41/W41
IL-7Rα
-/-
tg bcl-2
IL-7Rα
-/-
Kit
W41/W41
tg bcl-2

Figure 4.1. Lack of cortical and medullary regions in IL-7
-/-
Kit
W41/W41

and IL-7Rα
-/-
Kit
W41/W41
mice. Thymus sections were stained for H&E
stains. Magnification: 100x.
IL-7Rα
-/-
Kit
W41/W41

59

Figure 4.2. TEC differentiation block in IL-7Ra
-/-
Kit
W41/W41
thymus and relieve of
the differentiation block by enforced bcl-2 expression. Cytokeratin (K)
expression analysis of thymuses. Thymus sections were stained with anti-
cytokeratin 5 and anti-cytokeratin 8 and subsequently with secondary antibodies.
Magnification: 200x.

K
8
K
5
Wild
type
Kit
W41/W41

IL-7
-/-

IL-7Ra
-/-

K8

K5

K8/K5
IL-7Ra
-/-

tg bcl-2
IL-7Ra
-/-

Kit
W41/W41

IL-7Ra
-/-

Kit
W41/W41

tg

bcl-2
IL-7
-/-

Kit
W41/W4
1

60
4.5 Discussion

Thymic crosstalk between developing thymocytes and thymic epithelial cells
previously was thought to be essential for normal thymic structural development.
However, recent studies have shown that there seems to be no absolute
requirement for the crosstalk for the proper thymic development. It has been shown
that the three-dimensional architecture of the thymus is required to maintain Delta-
like expression necessary for inducing T cell development (Mohtashami 2006).
Thymuses of the doubly knockout mice examined in this study lack medullary or
cortical areas as well as three-dimensional structures indicated by the absence of
K8+K5- or K8-K5+ TECs and their flat disk-like structure. These thymuses are small
compared to those of wild type or parental mutant strains.
Critical roles of thymic epithelial cells (TEC) in supporting normal
development of thymic architecture have been elucidated (Klug 2002). Specific
interaction of thymocytes and thymic epithelial cells which occurs in the cortex and
medulla of the thymus is indispensable in thymocyte differentiation and proper
development of three-dimensional cortical and medullary structure (Hollander 1995).
Differentiation stages of thymic epithelial cells can be phenotypically defined by
cytokeratin 5 (K5) and cytokeratin 8 (K8) expression pattern (Klug 2002). Our results
indicate that the thymuses of IL-7
−/−
Kit
W41/W41
and IL-7Rα
−/−
Kit
W41/W41
mice
predominantly consist of immature K5 and K8 double-positive thymic epithelial cells
whereas thymuses of wild type and parental strains have distinctive regions of more
differentiated K5 or K8 single positive cells. This suggests that in the IL-7
−/−
Kit
W41/W41

61
and IL-7Rα
−/−
Kit
W41/W41
thymuses which consist mostly of Thy-1-negative non-T
lineage cells the lack of normal thymocyte differentiation results in an abnormal,
immature pattern of thymic epithelial cell differentiation. H&E staining of the
thymuses of these mice have an appearance similar to that of tgε26 mice in which
human CD3ε is over-expressed and thymocyte differentiation is blocked at DNI
(CD44+CD25-) stage, with absence of a three-dimensional structure, presence of
pale staining epithelial cells and a small number of thymocytes (Hollander 1995).
The transition from immature TEC phenotypes, such as co-expression of K5 and K8,
to the fully differentiated medulla-cortex organization is perturbed in mice in which T
cell development is blocked at immature stages (Rodewald 2008). Large clusters of
immature K5+K8+ TEC are maintained in mutants with massive early blockade in T
cell development such as Kit
W/W
γc− (Rodewald 1998), or RAG2−/−γc− mice (Gill
2003).
Previous studies have shown that thymic development can be classified into
three phases based on the interaction of thymocytes and thymic epithelial cells:
thymocyte independent differentiation, establishment of three-dimensional cortical
architecture, and establishment of organized medulla (Hollander 1995). Analyses of
IL-7
−/−
Kit
W41/W41
and IL-7Rα
−/−
Kit
W41/W41
thymuses for K5 and K8 expression have
shown that these thymuses lack organized cortical or medullary regions which is
consistent with the block in thymocyte differentiation at CD44+CD25- DN stage I in
these mice. Predominance of K5 and K8 double positive immature TECs in these
thymuses indicates that because of the block in differentiation of immature
thymocytes in IL-7
−/−
Kit
W41/W41
and IL-7Rα
−/−
Kit
W41/W41
mice at DN1 (CD44+CD25-)

62
proper development of TECs does not occur, and this results in the absence of K5
or K8 single positive medulla or cortex, respectively. Whereas the thymuses of IL-
7Rα
−/−
Kit
W41/W41
are hypocellular with a small number of thymocytes and are
predominantly K5+K8+, thymuses of IL-7Rα
−/−
Kit
W41/W41
tg bcl-2 mice have areas of
hypercellularity, and these areas are K5+K8- indicating that medullary regions
developed in the thymuses. These mice have a higher cellularity and more
differentiated thymocytes including CD4+ and CD8+ cells than mice without the bcl-
2 transgene. The emergence of K5+K8- medullary regions in these mice is
consistent with the idea of crosstalk between TECs and thymocytes and the
requirement of developing thymocytes and their proper differentiation in order for
normal TEC differentiation and development of cortex and medulla to occur
(Hollander 1995). Even the small increase in thymocyte number (250 to 2x10
4
; wild
type, 114x10
6
) resulted in the emergence of K5+K8- medullary regions in the
thymuses of IL-7Rα
−/−
Kit
W41/W41
tg bcl-2 mice.

Conclusion and Future Directions
This study has shown that IL-7R and c-kit signaling is essential for
thymopoiesis and has synergistic, partially redundant, and unique roles in
thymopoiesis. Results from this study also suggest that c-kit provides more than
survival signals in thymopoiesis. A study by Rodewald et al. has shown that signals
transduced by γc and c-kit are essential for thymopoiesis (Rodewald 1997). It is now
shown that of the cytokines whose receptors utilize the γc, IL-7 provides signals that
are responsible for thymopoiesis. Data from the analysis of the results of enforced

63
expression of a bcl-2 transgene has shown that c-kit may provide instructive signals
which results in differentiation of immature DN thymocytes.
More detailed elucidation in the nature of signaling pathways downstream of
IL-7R and c-kit and interactions between them will facilitate further understanding of
the process of thymopoiesis. For example, synergism between IL-7R and c-kit which
is observed in thymopoiesis may be due to the interaction of the two receptors and
production of quantitative (persistent phosphorylation of STAT5) or qualitative
(STAT heterodimer production) differences compared to the results of signaling
through either receptor alone (Greenlund 1995). Determination of these differences
in thymocytes of parental strains and doubly mutated mice will likely provide
molecular bases of the phenotypic differences between them.
A number of putative circulating and thymus seeding T-lineage progenitors
have been identified. Further elucidation of the nature of these progenitors which
are physiologically important will facilitate understanding of the process of
thymopoiesis. Similarly, elucidation of the nature and function of thymic niches
which are required for thymocyte development and eventual production of T cells
will provide useful information of potentially clinical significance.
Damage to TEC due to aging, graft-versus host disease, and pre-
hematopoietic stem cell transplantation (HSCT) regimen such as chemotherapy and
irradiation is associated with decreased thymopoiesis and subsequent immune
deficiency. It has been shown that post-HSCT thymopoietic defect can be corrected
by administration of IL-7, resulting in restoration of normal thymus cellularity,
thymocyte maturation, increase in mature T cells and antigen-specific responses

64
(Bolotin 1996). Elucidation of detailed mechanism by which thymocytes and TEC
exert mutual influence in thymus function and reconstitution after HSCT will likely
have significant clinical implication and value.

65
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Abstract (if available)

Abstract IL-7 and Kit ligand (KL) are cytokines produced by thymic epithelial cells, which interact with their cognate receptors on immature thymocytes. The IL-7R is comprised of the IL-7Rα and common γ chain (γc) and has no intrinsic kinase activity, while KL binds to the receptor tyrosine kinase Kit. Both IL-7Rα^-/- and IL-7^-/- mice have profound defects in thymopoiesis, although for unexplained reasons, the defects in differentiation and thymic cellularity are more severe for IL-7Rα -/- than IL-7-/- mice. In order to understand possible interactions between IL-7R and Kit signaling in vivo, we generated doubly mutated mice which were homozygous for the Kit^W41 loss of function mutation and null for either IL-7 or IL-7Rα. While IL-7^-/- and IL-7Rα^-/- mice had a 90-99% reduction in thymic cellularity and the Kit^W41/W41 mice had a 50% reduction, the IL-7-/-Kit^W41/W41 and IL-7Rα-/-Kit^W41/W41 mice had fewer than 200 thymocytes, representing a 5-6 log decrease in thymic cellularity. The thymocytes in the IL-7^-/-Kit^W41/W41 and IL-7Rα^-/-Kit^W41/W41 mice were blocked at the earliest recognizable stage of thymic differentiation. The frequency of early Tlineage progenitors (ETP) in IL-7Rα^-/-, IL-7^-/-Kit^W41/W41, and IL-7Rα^-/-Kit^W41/W41 mice was significantly reduced compared to parental strains or wild type mice. Introduction of a bcl-2 transgene did not relieve the block in differentiation of CD4- CD8- (DN) thymocytes, or reduction in ETP absolute numbers in IL-7Rα^-/-Kit^W41/W41 mice, but partially rescued IL-7Rα^-/- mice. Cytokeratin expression analysis showed that thymic epithelial cells (TEC) of IL-7^-/-Kit^W41/W41, and IL-7Rα^-/-Kit^W41/W41 mice were K8+K5+, indicating that differentiation of TEC was arrested in these mice. IL-7Rα^-/- Kit^W41/W41 transgenic bcl-2 thymuses had K8+K5- areas indicating that medullary areas developed. Conclusions: 1) IL-7R and Kit provide synergistic, partially redundant, and unique signals for thymocyte proliferation, ma

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Creator Toyama, Akira (author)

Core Title IL-7R and c-Kit signaling in thymopoiesis

School School of Dentistry

Degree Doctor of Philosophy

Degree Program Craniofacial Biology

Publication Date 12/04/2008

Defense Date 10/22/2008

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag cytokine signaling,OAI-PMH Harvest,thymopoiesis,T-lineage progenitors

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Lutzko, Carolyn (committee chair), Crooks, Gay M. (committee member), Le, Anh D. (committee member), Paine, Michael L. (committee member), Shi, Songtao (committee member)

Creator Email atoyama@usc.edu,rz350lc@dslextreme.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m1872

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Rights Toyama, Akira

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