T cell Development

LSK, Flt3-

HSC

Flt3low
MPP
Haematopoiesis

cKit+Sca-1- Lin- Rag+/il7ra+

Ly6D-
CD34+ Flt3high

CMP LMPP/ ELP ALP

Ly6D+

+CD32/16high +CD32/16low BLP

Lin-Sca-1lowcKitlow,il7r+
GMP MEP CLP
Bone marrow environment
T cell development occurs in the thymus

4
The Thymus is required for T cell development
scid mutation: nude mutation:
DNA PK transcription
no gene factor required for
rearrangement terminal epithelial
differentiation
(whn)

NO B or T CELLS

5
Thymus structure

6
 T cell development
The precursors in the bone marrow that leave the bone marrow and migrate to the thymus are destined to become T
cells.
Recruitment of adult thymic progenitors is regulated by P-selectin and its ligand PSGL-1
the precursor, which is directed to the thymus via chemokine receptors, retains the potential to give rise to more than one
type of cell, including natural killer (NK) cells, dendritic cells (DC), B cells, and even myeloid cells. This precursor only
becomes fully committed to the T-cell lineage in the late DN2 stage of T-cell development
The Ly6d− subset, called ALP (all-lymphoid progenitor), retains full lymphoid potential and early thymic seeding
activity, whereas the Ly6d+ subset, called BLP (B-cell-biased lymphoid progenitor), up-regulates the B-cell-specifying
factors Ebf1 and Pax5 and behaves essentially as a B-cell progenitor. Thus, Ly6d identifies the first stage of B-cell
development (BLP), and the population residing at the branch point between B- and T-cell development (ALP).
Notch target genes that encode molecules critical for T-cell commitment include the transcription factors T-cell factor-7
(TCF-7). Another transcription factor regulated by Notch signaling and necessary for T-cell development is GATA-
binding protein 3 (GATA-3). GATA-3 is a zinc-finger transcription factor that is required during multiple stages of T-cell
development, as well as in T-cell function. GATA-3 is expressed as early as the ETP stage and is critical for the
development of this cell population.

* Why some T cell precursors migrate from bone marrow to thymus:

 Notch signalling leads to T cell development
commitment to the T-cell lineage was dependent on a receptor, Notch, which
had been classically associated with embryonic cell development. Notch, in
fact, regulates the decision of a lymphoid precursor to become a T versus a B
lymphocyte. When a constitutively active version of Notch1, one of four
versions of Notch, is overexpressed in hematopoietic cells, T cells rather than
B cells develop in the bone marrow. Reciprocally, when the Notch1 gene is
knocked out among hematopoietic precursors, B cells rather than T cells
develop in the thymus.
Investigators can induce lymphoid development from hematopoietic stem
cells in vitro using a combination of stromal cell lines and soluble cytokines
and growth factors, as indicated. Investigators discovered that Notch
signaling was the key to inducing development to the T- rather than B-
lymphocyte lineage. After transfecting the stromal cell line with a gene
encoding the Notch ligand, lymphoid precursors would adopt the T-cell
lineage. Otherwise, they would develop into B cells.
Cells in the thymus

9
T cell receptor complex
 Thymocytes Progress through Four Double-Negative Stages

T-cell development is elegantly organized, spatially and temporally. Different stages of development take place in distinct
microenvironments that provide membrane-bound and soluble signals that regulate maturation. After arriving in the thymus
from the bone marrow via blood vessels at the cortico-medullary boundary, T-cell precursors encounter Notch ligands, which
are abundantly expressed by the thymic epithelium. T-cell precursors first travel to the outer cortex where they slowly
proliferate, then they pass through the thymic medulla before exiting at the corticomedullary junction. During the time it
takes cells to develop in the thymus (1 to 3 weeks), thymocytes pass through a series of stages defined by changes in their
cell surface phenotype. The earliest T cells lack detectable CD4 and CD8 and are therefore referred to as double-negative
(DN) cells. DN T cells can be subdivided into four subsets (DN1-4) based on the presence or absence of other cell surface
molecules, including c-kit (CD117), the receptor for stem cell growth factor; CD44, an adhesion molecule; and CD25, the 
chain of the IL-2 receptor.
Structure of Thymus
The T cell developmental events take place in several distinct thymic
microenvironments. T-cell precursors enter the thymus in blood vessels at
the corticomedullary junction between the thymic cortex, the outer portion
of the organ, and the thymic medulla, the inner portion of the organ. At this
stage thymocytes express neither CD4 nor,CD8, markers associated with
mature T cells. They are therefore called double negative (DN) cells. DN
cells first travel to the region under the thymic capsule, a region referred to
as the subcapsular cortex, where they proliferate and begin to generate their
T-cell receptors. Thymocytes that successfully express TCRs begin to
express both CD4 and CD8, becoming double positive (DP) cells, and
populate the cortex, the site where most (85% or more) immature T cells
are found. The cortex features a distinct set of stromal cells, cortical thymic
epithelial cells (cTECs), whose long processes are perused by thymocytes
testing the ability of their T-cell receptors to bind MHC-peptide complexes.
Thymocytes that survive selection move to the thymic medulla, where
positively selected thymocytes encounter specialized stromal cells,
medullary thymic epithelial cells (mTECs). Not only do mTECs support
the fi nal steps of thymocyte maturation, but they also have a unique ability
to express proteins that are otherwise found exclusively in other organs.
This allows them to negatively select a group of potentially very damaging,
autoreactive T cells that could not be deleted in the cortex. Mature
thymocytes, which express only CD4 or CD8 and are referred to as single
positive (SP), leave the thymus as they entered: via the blood vessels of the
corticomedullary junction. Maturation is finalized in the periphery, where
these new T cells (recent thymic emigrants) explore antigens presented in
secondary lymphoid tissue, including spleen and lymph nodes.
cKit
receptor tyrosine kinase, c-kit is an essential molecule for constitutive intra-marrow hemopoiesis, especially
for the self-renewal of hemopoietic progenitor cells at various stages of differentiation.

CD44
CD44 has been shown to regulate HSC and their BM microenvironment by influencing: 1) matrix assembly; 2)
cytokine/chemokine capture and/or release; 3) cytoskeletal linker protein binding (eg. ankyrin, ezrin, radixin and
moesin) and signal transduction; and 4) matrix degradation via protease production to influence HSC adhesion,
homing, migration, quiescence, resistance to oxidative stress as well as mobilization

CD25
CD25, also known as Cluster of Differentiation 25, is an essential molecule involved in immune responses. It is the
alpha chain of the high-affinity interleukin-2 receptor (IL2RA). This 65kDa transmembrane glycoprotein is primarily
located on the cell surface of activated lymphocytes, playing a crucial role in their proliferation, activation.
 Stages of T cell development in thymus

Directional migration of developing cells through distinct

thymic microenvironments is essential for proper T cell
maturation, since this migration allows immature
thymocytes to receive differentiation signals transmitted
by different sets of epithelial cells that are located at
distinct cortical regions. Interestingly, recent studies have
shown that the generation and maintenance of intact
cortical microenvironments also requires interaction of the
stromal cells with developing thymocytes, establishing a
feedback mechanism called “thymic cross talk”.
Interaction of migrating precursors with stromal cells
appears to be constitutive because the latter cells express
counter-receptors for progenitor adhesion molecules and
serve as a matrix for their migration. The molecular
mechanisms that govern intrathymic migration are poorly
understood. Although several studies suggested that
chemokines and their receptors are involved in this
process, the role of these molecules during T cell
maturation in vivo has been only partially elucidated. A
recent study has shown that signaling through CXCR4 is
crucial for the entry of thymus-homing progenitors into
the cortex, and consequently the development of cells
lacking CXCR4 is arrested at the DN1 stage.
Different stages of T cell development in the Thymus
cKit: Stem cell factor receptor
CD44: hyaluronan (HA) and fibronectin (FN)
CD25: a chain of IL2 receptor

For human, The earliest thymic progenitors are

CD34+CD38−CD1a−, followed by
CD34+CD38+CD1a− and
CD34+CD38+CD1a+ cells

15
DN1 thymocytes are the first to enter the thymus and are still capable of giving rise to multiple cell types. They express
only c-kit and CD44 (c-kit++CD44+CD25- )but once they encounter the thymic environment and become resident in the
cortex, they proliferate and express CD25, becoming DN2 thymocytes (c-kit++CD44+CD25+ ). During this critical stage of
development, the genes for the TCR ,  and  chains begin to rearrange; however, the TCR  locus does not rearrange,
presumably because the region of DNA encoding TCR  genes is not yet accessible to the recombinase machinery. At the
late DN2 stage, T-cell precursors fully commit to the T-cell lineage and reduce expression of both c-kit and CD44.
Cells in transition from the DN2 to DN3 (c-kit+CD44-CD25+ ) stages continue rearrangement of the TCR  
and TCR  chains and make the first major decision in T-cell development: whether to join the TCR  or TCR  T-cell
lineage.
Vertebrates generate two broad categories of T cells: those that express TCR  and  receptor chains and those
that express TCR  and  receptor chains. TCR  cells are the dominant participants in the adaptive immune response in
secondary lymphoid organs; however, TCR  cells also play an important role, particularly in protecting our mucosal
tissues from outside infection. Those DN3 T cells that successfully rearrange their  chain and therefore commit to the
TCR  T-cell lineage lose expression of CD25, halt proliferation, and enter the final phase of their DN stage of
development, DN4 (c-kitlow/-CD44-CD25- ), which mature directly into CD4+CD8+ DP thymocytes.

Why  T cells are dominant?

TCR genes are generated by the shuffling (rearrangement) of V and J (and sometimes D) segments, an event responsible
for the vast diversity of receptor specificities. Rearrangement of the   and  loci begins during the DN2 stage. To
become an  T cell, a cell must generate a TCR  chain—an event that depends on a single in-frame VDJ
rearrangement event. To become a  cell, however, a thymocyte must generate two functional proteins that depend on two
separate in-frame rearrangement events. Probability favors the former fate and, in fact, T cells are at least three times as
likely to become TCR  cells than TCR  cells.
 Difference between  T cells and  T cells
TCR  T-cell generation is also regulated developmentally. They are the first T cells that arise during fetal development, and
provide a very important protective function perhaps even prior to birth. Studies show, for instance, that  T cells are
required to protect very young mice against the protozoal pathogen that causes coccidiosis. However, production of  T cells
declines after birth, and the TCR T-cell population represents only 0.5% of all mature thymocytes in the periphery of an adult
animal. Most TCR  T cells are quite distinct in phenotype and function from conventional TCR  T cells. Most do not go
through the DP stage of thymocyte development and leave the thymus as mature DN T cells. Many emerge from the thymus
with the ability to secrete cytokines, a capacity gained by most TCR  cells only after they encounter antigen in secondary
lymphoid tissues. The TCR  T-cell population also expresses receptors that are not as diverse as TCR  T cells, and many
appear to recognize unconventional antigens, including lipids associated with unconventional MHC molecules. Many take up
long-term residence in mucosal tissues and skin and join innate immune cells in providing a first line of attack against
invading microbes, as well as the response to cellular stress.
DN1 and DN2 thymocytes do not express any T-cell receptor proteins on their surface. The pre-TCR is assembled during the transition from
the DN2 to the DN3 stage of development, when a successfully rearranged TCR β chain dimerizes with the nonvariant pre-Tα chain. Like
the mature αβ TCR dimer, the pre-TCR is noncovalently associated with the CD3 complex. Successful assembly of this complex results in
intracellular signals at the DN3 stage that induce a variety of processes, including the maturation to the DP stage and rearrangement of the
TCR α chain. Once a thymocyte has successfully rearranged a TCR α chain (in transition between the DN4 and DP stages), this chain
dimerizes with the TCR β chain, replacing the pre-Tα chain and generating a mature αβ TCR. The αβ TCR expressed by DP thymocytes also
complexes with CD3 and can generate signals that lead to either positive or negative selection (differentiation or death, respectively),
depending on the affinity of its interaction with the MHC/peptide complexes it encounters in the thymic cortex and medulla. Although the αβ
TCR/CD3 complex expressed by mature SP T cells is structurally the same as that expressed by DP thymocytes, the signals it generates are
distinct. It responds to high-affinity engagement not by dying, but by initiating cell proliferation, activation, and the expression of effector
functions. Low-affinity signals generate survival signals. The basis for the differences in consequence of signals generated by DP and SP
TCR complexes is still unknown.
  selection
Double-negative (DN) thymocytes that have successfully rearranged their TCR  chains are valuable, and are identified and
expanded via a process known as  selection. This process involves a protein that is uniquely expressed at this stage of
development, a 33-kDa invariant glycoprotein known as the pre–T  chain. Pre-T acts as a surrogate for the real TCR 
chain, which has yet to rearrange, and assembles with a successfully rearranged and translated  chain, as well as CD3
complex proteins. This precursor TCR/CD3 complex is known as the pre-TCR and acts as a sensor by initiating a signal
transduction pathway. The signaling that the pre-TCR complex initiates is dependent on many of the same T-cell specific
kinases used by a mature TCR, but does not appear to be dependent on ligand binding. In fact, little if any of the complex is
expressed on the cell surface; rather, successful assembly of the complex may be sufficient to activate the signaling events.
The Pre-TCR leads to the following events:
1. Maturation to the DN4 stage (c-kitlow/-CD44-CD25-)
2. Rapid proliferation in the subcapsular cortex
3. Suppression of further rearrangement of TCR -chain genes,
resulting in allelic exclusion of the -chain locus
4. Development to the CD4+CD8+ double-positive (DP) stage
5. Cessation of proliferation
6. Initiation of TCR  chain rearrangement
It is important to note that the proliferative phase prior to  chain rearrangement enhances receptor diversity
considerably by generating clones of cells with the same TCR -chain rearrangement. Each of the cells within a clone
can then rearrange a different  -chain gene, thereby generating an even more diverse population than if the original cell
had undergone rearrangement at both the  - and -chain loci prior to proliferation. TCR -chain gene rearrangement
does not begin until double-positive thymocytes stop proliferating.

Allelic Exclusion
Most T cells fully rearrange and express a TCR  chain from only one of their two TCR alleles, a phenomenon known as
allelic exclusion. Allelic exclusion is the result of inhibition of further rearrangement at the other TCR allele (which
must be fully rearranged to be expressed). This can be accomplished by reducing RAG expression so no more
rearrangement can occur, as well as by making the locus inaccessible to further RAG interaction via more permanent
changes in chromatin packaging. The details of the mechanisms responsible for this shutdown are still being investigated.
However, negative feedback signals from a successfully assembled pre-TCR/pre-T  complex during -selection clearly
have a significant influence. Other events, including the proliferative burst that follows -selection, which dilutes RAG
protein levels, can also play a role. RAG levels continue to change after -selection. They are restored after the
proliferative burst and allow TCR  rearrangement to occur. They decrease once again after expression of a successfully
assembled TCR  dimer.
Once a young double-positive (DP) thymocyte successfully rearranges and expresses a TCR  chain, this chain will
associate with the already produced TCR  chain, taking the place of the surrogate pre-TCR  chain, which is no longer
actively expressed. At this point, several “goals” have been accomplished by the thymus: hematopoietic cell precursors
have expanded in the subcapsular cortex, committed to the T-cell lineage, and rearranged a set of TCR genes. They have
also “chosen” to become a TCR  or TCR T cell. This TCR  population now expresses both CD4 and CD8, and is
ready for the second stage of T-cell development, that is selection.
 Positive selection & Negative Selection
CD4+CD8+(DP) thymocytes, small, nonproliferating cells that reside in the thymic cortex, are the most abundant subpopulation in the
thymus, comprising more than 80% of cells. They are also the first subpopulation of thymocytes that express a fully mature surface TCR
/CD3 complex and are therefore the primary targets of thymic selection. Thymic selection shapes the TCR repertoire of DP thymocytes
based on the affinity of their T-cell receptors for the MHC/peptides they encounter as they browse the thymic cortex.
Thymic selection is necessary because most distinctive property of the mature T cells is that they recognize only foreign antigen combined
with self-MHC molecules. However, randomly generated TCRs will certainly have no inherent affinity for “foreign antigen plus self-MHC
molecules.” They could just as well recognize foreign MHC/peptide combinations, which would not be useful, or self-MHC/self-peptide
combinations, which could be dangerous.

Two distinct selection processes are required:

• Positive selection: selects for those thymocytes bearing receptors capable of binding self-MHC molecules, resulting in MHC restriction
• Negative selection: selects against thymocytes bearing high-affinity receptors for self-MHC/peptide complexes, resulting in self-
tolerance.
Because only self-peptides are presented in the thymus, and only in association with self-MHC
molecules, these two selection processes ensure that surviving thymocytes express TCRs that have
low affinity for self-peptides in self-MHC. On the other hand, the processes do not guarantee that the
T cells generated will bear receptors with high affinity for any specific self-MHC/foreign peptide
combination.
The vast majority of DP thymocytes (~98%) never meet the selection criteria and die by
apoptosis within the thymus. The bulk of DP thymocyte death (~95%) occurs among thymocytes that
fail positive selection because their receptors do not specifically recognize self-MHC molecules.
These cells do not receive survival signals through their TCRs, and die by a process known as death
by neglect. A small percentage of cells (2%–5%) are eliminated by negative selection. Only 2% to 5%
of DP thymocytes actually exit the thymus as mature T cells.
 Zinkernagel and Dorothy experiment
He and his colleagues were curious to know how T cells became so “restricted.” They
considered two possibilities: Either the T cell and APC simply had to have matching
MHC types (i.e., an “A” strain T cell had to see an “A” strain antigen presenting cell) or T
cells, regardless of their own MHC type, “learned” the MHC type of their host sometime
during development. Zinkernagel and colleagues thought that such learning could take
place in the thymus, the T-cell nursery. To determine if T cells could be “taught” to
recognize the host MHC, they removed the thymus (thymectomized) and irradiated (A x
B) F1 mice so they had no functional immune system. They then reconstituted the
hematopoietic cells with an intravenous infusion of F1 bone marrow cells, but replaced
the thymus with one from a B-type mouse. (To be certain that the thymus graft did not
contain any mature T cells, they irradiated it before transplantation.)
In this experimental system, T-cell progenitors from the (A x B) F1 bone marrow would
mature within a thymus that expresses only B-haplotype MHC molecules on its stromal
cells. Would these (A x B) F1 T cells now be MHC restricted to the B haplotype of the
thymus in which they developed? Or, because they expressed both A and B MHC, would
they be able to recognize both A and B MHC haplotypes? To answer this question, the
investigators infected the chimeric mice with lymphocytic choriomeningitis virus (LCMV,
the antigen) and removed the immunized, mature splenic T cells to see which LCMV-
infected target cells (APCs) they could kill. They tested them against infected APCs from
strain A, strain B, and strain A B mice. T cells from the chimeric mice could only lyse
LCMV infected target cells from strain B mice. Thus, the MHC haplotype of the thymus
in which T cells develop determines their MHC restriction. T cells “learned” which MHC
haplotype they are restricted to during their early days in the thymus.
 Positive selection
In the thymus, thymocytes come into contact with thymic epithelial cells that
express high levels of class I and class II MHC molecules on their surface. These
self-MHC molecules present self-peptides, which are typically derived from
intracellular or extracellular proteins that are degraded in the normal course of
cellular metabolism.
Thymic stromal cells, including epithelial cells, macrophages, and dendritic
cells, play essential roles in positive and negative selection and should be part of
our visualization of thymic selection events. Young DP thymocytes are in
intimate contact with these cells and “browse” the MHC/self-peptides displayed
on their surfaces. Each of these cell types has the capacity to express high levels
of class I and class II MHC proteins, and typically have extended processes that
contact many developing thymocytes. Some also express costimulatory ligands,
including CD80 (B7-1) and CD86 (B7-2). As the thymocytes migrate through
the thymus, they encounter multiple different stromal cell surfaces, and have the
opportunity to bind many different MHC/peptide combinations.
DP thymocytes can be considered to have one of three fates, depending on the
affinity of their new T-cell receptors for the MHC/self peptide combinations that
they encounter: If their newly generated TCRs do not bind to any of the
MHC/self-peptides they encounter on stromal cells, they will die by neglect. If
they bind too strongly to MHC/self-peptide complexes they encounter, they will
be negatively selected. If they bind with a low, “just right” affinity to MHC/self-
peptide complexes, they will be positively selected and mature to the single
positive stage of development.
 Autoreactive CD4+CD8+ thymocytes with high-affinity receptors for self-MHC/self-peptide
Negative selection
combinations are potentially dangerous to an organism, and many are killed by negative
selection in the thymus. In fact, errors in the negative selection process are responsible for a
host of autoimmune disorders, including Type 1 diabetes. Negative selection is defined
broadly as any process that rids a repertoire of autoreactive clones and is responsible for
central tolerance. It is likely that most negative selection occurs via a process known as
clonal deletion, where high-affinity TCR interactions directly induce apoptotic signals.
Clonal deletion of DP thymocytes appears to be optimally mediated by the same cells
(APCs) and same interactions (high-affinity TCR engagement coupled with costimulatory
signals) that activate mature T cells. Why strong TCR signals result in death of immature T
cells, but proliferation and differentiation of mature T cells remains an active area of
investigation.
Thymic dendritic cells and macrophages, which are found in multiple areas of the thymus,
clearly have the ideal features to mediate negative selection, but interestingly, so do
medullary epithelial cells, which express high levels of the costimulatory ligands CD80 and
CD86 as well as a unique transcription factor called Auto immune regulator (AIRE) that
allows them to present tissue-specific antigens. AIRE’s mechanism of action is still under
investigation. It has classic features of a transcription factor and may be part of a
transcriptional complex that facilitates expression of tissue-specific genes by regulating not
only translation, but also chromatin packing. Thus, it allows medullary epithelial cells to
express proteins not ordinarily found in the thymus, process them, and present them in MHC
molecules. This particularly useful for the presentation of MHC Class I peptides and
negative selection of CD8+ thymocytes. However, neighboring dendritic cells and
macrophages are also thought to phagocytose medullary epithelial cells and so can then
present their protein contents in MHC Class II molecules, and mediate negative selection of
CD4+ thymocytes
MHC Expression
 Single positive cell generation
affinity for self-antigens, they are also being guided in their lineage decisions. Specifically, a
positively selected double positive thymocyte must decide whether to join the CD8+ cytotoxic T-cell
lineage or the CD4 helper T-cell lineage. Lineage commitment requires changes in genomic
organization and gene expression that result in (1) silencing of one coreceptor gene (CD4 or CD8) as
well as (2) expression of genes associated with a specific lineage function.
Instructive model: TCR/CD4 and TCR/CD8 coengagement generates unique signals that directly
initiate distinct developmental programs. For example, if a thymocyte randomly generated a TCR
with an affinity for MHC Class I, the TCR and CD8 would bind MHC Class I together, and generate
a signal that specifi cally initiated a program that silenced CD4 expression and induced expression of
genes specific for cytotoxic T-cell lineage function. Likewise, TCR/CD4 coengagement would
generate a unique signal that initiated CD8 silencing and the helper T-cell developmental program.
stochastic model: a positively selected thymocyte randomly down-regulates CD4 or CD8. Only
those cells that express the “correct” coreceptor—the ones that can coengage MHC with the TCR—
generate a TCR signal strong enough to survive to mature. In this model, TCR/CD4 and TCR/CD8
coengagement does not necessarily generate distinct signals. Unfortunately, studies that followed the
consequences of such mismatches confounded researchers by providing evidence in support of both
models! Clearly they were too simplistic.
kinetic signaling model: It propose that thymocytes commit to the CD4+ T-cell lineage if they
receive a continuous signal in response to TCR/coreceptor engagement, but commit to the CD8
lineage if the TCR signal is interrupted. We now know that all CD4+CD8+ thymocytes down-
regulate surface levels of CD8 in response to positive selection. Given this response, only MHC
Class II restricted T cells will maintain continuous TCR/CD4/MHC Class II interaction, and
therefore develop to the CD4 lineage. However, with the loss of CD8 expression, MHC Class I
restricted T cells will lose the ability to maintain TCR/CD8/MHC Class I interactions. Singer et al.
provide evidence that these thymocytes, interrupted from their TCR/ coreceptor engagement, are
subsequently rescued by IL-7, which facilitates their commitment to the CD8+ lineage.
T cell exit from Thymus

KLF2 is a transcription factor that is

Responsible for the upregulation of
S1PR1.
 Recent Thymic Emigrants Cells

T cell maturation was once thought to occur entirely within the thymus. Now, evidence is mounting that the youngest
peripheral T cells in both mice and humans comprise a distinct population from their more mature, yet still naive,
counterparts. These cells, termed recent thymic emigrants (RTEs), undergo a process of post-thymic maturation that can be
monitored at the levels of cell phenotype and immune function.

Over a period of approximately 3 weeks, recent thymic emigrants (RTEs) undergo phenotypic maturation in the
lymphoid periphery. This is characterized by the downregulation of surface CD24 and T cell receptor (TCR)–CD3
expression and the upregulation of surface expression of CD45RB, QA2, interleukin-7 receptor α-chain (IL-7Rα) and
CD28.