REVIEWS

T-CELL DEVELOPMENT AND THE

CD4–CD8 LINEAGE DECISION
Ronald N. Germain
Cell-fate decisions are controlled typically by conserved receptors that interact with co-evolved
ligands. Therefore, the lineage-specific differentiation of immature CD4+CD8+ T cells into CD4+ or
CD8+ mature T cells is unusual in that it is regulated by clonally expressed, somatically generated
T-cell receptors (TCRs) of unpredictable fine specificity. Yet, each mature T cell generally retains
expression of the co-receptor molecule (CD4 or CD8) that has an MHC-binding property that
matches that of its TCR. Two models were proposed initially to explain this remarkable outcome
— ‘instruction’ of lineage choice by initial signalling events or ‘selection’ after a stochastic fate
decision that limits further development to cells with coordinated TCR and co-receptor
specificities. Aspects of both models now appear to be correct; mistake-prone instruction of
lineage choice precedes a subsequent selection step that filters out most incorrect decisions.

CO-RECEPTOR
A CD4 or CD8 molecule, which
cooperatively recognizes an
MHC class-II or class-I ligand,
respectively, together with the
antigen receptor (T-cell
receptor) of a T cell.
Lymphocyte Biology Section,
Laboratory of Immunology,
National Institute of Allergy
and Infectious Diseases,
National Institutes of
Health, Building 10,
Room 11N311 MSC-1892,
10 Center Drive,
Bethesda, Maryland
20892-1892, USA.
e-mail: rgermain@nih.gov
doi:10.1038/nri798
Cell-fate specification is a key developmental event.
The physiological function of a multicellular organism
depends on the generation of the proper number and
diversity of cell types. Signals from broadly expressed
receptors that interact with co-evolved germ-line lig-
ands control most differentiation decisions1. But, adap-
tive immunity depends on the function of T and B
cells, which express unique surface receptors that are
created by somatic DNA rearrangement and random
chain pairing. These clonal receptors help to determine
which precursor lymphocytes will successfully mature;
this raises the issue of whether these receptors dictate
cell fate in the same general manner as more broadly
expressed receptors or whether novel mechanisms
underlie their activity.
Such questions are particularly intriguing with
respect to the main peripheral pool of T cells that
express αβ T-cell receptors (TCRs). These cells
respond to antigen in the form of short peptides
bound to MHC class I or class II molecules2. MHC
class-I ligands alert the immune system to active intra-
cellular infection and target infected cells for destruc-
tion, whereas MHC class-II ligands guide intercellular
co-operation between haematopoietic cells in an
immune response and help to combat extracellular
infections. In accordance with this dichotomy, mature
αβ T cells comprise two lineages, the antigen recogni-
tion of which is focused on either MHC class-I- or
class-II-associated peptides and whose effector func-
tions are tuned to these recognition biases. Typically,
these subsets are defined by exclusive surface expression
of either the pan-MHC-class-II-binding protein CD4
(helper or regulatory CD4+ T cells) or the pan-MHC-
class-I-binding protein CD8 (cytotoxic CD8+ T cells)3,4.
CD4 and CD8 are non-clonally distributed proteins
that, because they co-recognize the ligands of the TCR,
are often referred to as CO-RECEPTORS5. Maintaining the
proper number and proportion of CD4+ and CD8+
T cells is essential for optimal host defence — the ques-
tion is how this required outcome can be achieved,
given the unpredictable peptide–MHC specificity of
each precursor cell’s TCR.
This problem was defined more precisely as the pre-
cursor–product relationships in αβ T-cell differentiation
were unravelled6 (FIG. 1). An important advance was the
recognition that CD4+ and CD8+ T cells are derived from
a common precursor pool, in which each cell expresses
both CD4 and CD8 proteins. Subsequent experiments7–10
indicated that the successful maturation of precursor
CD4+CD8+ thymocytes seems to be restricted to those

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CLONAL EXPRESSION
The presence of a particular
somatically generated antigen
receptor on a maturing T cell
that is not shared with other
independently developing
precursors. This receptor is
shared among the progeny of the
mature cell after activation and
cell division stimulated by
foreign antigen.
RECOMBINATION-ACTIVATING
GENE
(RAG). The product of this gene
is involved in creating the
double-strand DNA breaks that
are necessary for producing the
rearranged gene segments that
encode the complete protein
chains of T-cell and B-cell
receptors.
NEGATIVE SELECTION
Active cell loss in the thymus,
which is mediated by apoptosis
induced by strong T-cell stimuli,
particularly at later stages of
maturation.
POSITIVE SELECTION
The maturation of immature
CD4+CD8+ precursor
thymocytes induced by T-cell
receptor signals that result from
binding to self-peptide–MHC
ligands on thymic epithelial cells.
310 | MAY 2002 | VOLUME 2
Outer cortex
Inadequate
TCR/co-receptor Cortical
signalling epithelial
cell
MHC class I MHC class II
recognition recognition
Death by
Inner cortex
neglect
Lymphoid
progenitor
Cortico–medullary CD8-committed DP CD4-committed DP
junction
Negative Positive Negative
selection selection selection
Medulla
Haematopoietic
precursor
Medullary Death Death Medullary
epithelial cell/ epithelial cell/
dendritic cell dendritic cell
Emigration to periphery
Figure 1 | Overall scheme of T-cell development in the thymus. Committed lymphoid progenitors arise in the bone marrow and
migrate to the thymus. Early committed T cells lack expression of T-cell receptor (TCR), CD4 and CD8, and are termed double-negative
(DN; no CD4 or CD8) thymocytes. DN thymocytes can be further subdivided into four stages of differentiation (DN1, CD44+CD25−; DN2,
CD44+CD25+; DN3, CD44−CD25+; and DN4, CD44−CD25−)41. As cells progress through the DN2 to DN4 stages, they express the pre-
TCR, which is composed of the non-rearranging pre-Tα chain and a rearranged TCR β-chain43. Successful pre-TCR expression leads
to substantial cell proliferation during the DN4 to double positive (DP) transition and replacement of the pre-TCR α-chain with a newly
rearranged TCR α-chain, which yields a complete αβ TCR. The αβ-TCR+CD4+CD8+ (DP) thymocytes then interact with cortical epithelial
cells that express a high density of MHC class I and class II molecules associated with self-peptides. The fate of the DP thymocytes
depends on signalling that is mediated by interaction of the TCR with these self-peptide–MHC ligands6,51. Too little signalling results in
delayed apoptosis (death by neglect). Too much signalling can promote acute apoptosis (negative selection); this is most common in the
medulla on encounter with strongly activating self-ligands on haematopoietic cells, particularly dendritic cells123. The appropriate,
intermediate level of TCR signalling initiates effective maturation (positive selection). Thymocytes that express TCRs that bind self-
peptide–MHC-class-I complexes become CD8+ T cells, whereas those that express TCRs that bind self-peptide–MHC-class-II ligands
become CD4+ T cells; these cells are then ready for export from the medulla to peripheral lymphoid sites. SP, single positive.
cells that maintain expression of the co-receptor molecule instruction12,13, then to selection14–19, and more recently,
(CD4 or CD8) that has a self-MHC-class specificity that back to instruction20–36. This review summarizes our
matches the CLONALLY EXPRESSED TCR. There are two possi- current understanding of αβ T-cell differentiation,
ble explanations for this phenomenon: either the interac- antigen-receptor signalling and the TCR–co-receptor-
tion of a TCR–co-receptor pair with an MHC ligand dependent events that guide the lineage-specific devel-
instructs the cell as to which path to take and which opment of immature T cells to yield pools of CD4+ and
(inappropriate) co-receptor-encoding gene to stop tran- CD8+ mature T cells that are properly suited to their
scribing; or, after random (stochastic) lineage choice and physiological roles.
the extinction of CD4 or CD8 expression, further matu-
ration depends on signals that require a match between The αβ T-cell differentiation pathway
the MHC-class specificity of the remaining co-receptor Generation of CD4+CD8+ immature thymocytes.
and the cell’s TCR (selection11,12) (FIG. 2). Committed lymphoid progenitors arise in the bone
Over the years, the prevailing view has swung back marrow and migrate via the blood to the thymus. In the
and forth between these two possibilities — first to thymus, these cells lose the potential for B-cell37–39 and
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 REVIEWS

a Instruction b Stochastic/selection natural-killer-cell40 development. The result is a double-

negative (DN; no CD4 or CD8), committed T-cell pre-
TCR–CD8/ TCR–CD4/ Stochastic choice of cursor (FIG. 1). DN thymocytes can be subdivided further
MHC class I MHC class II CD4 or CD8 lineage
interaction interaction (before or at time into four sequential stages of differentiation, which are
of first TCR signal) identified by their surface expression of CD44 and
Cortical
epithelial cell CD25: DN1, CD44+CD25−; DN2, CD44+CD25+; DN3,
CD44−CD25+; and DN4, CD44−CD25− (REF. 41).
Correct
CD8
Incorrect Double-negative T cells can give rise to either γδ or
CD4
MHC CD8 MHC
choice
choice αβ TCR-expressing cells6. For cells that proceed along
class I class II the αβ TCR pathway, DN3-stage cells first express pre-
TCR CD4 TCR-α42–44, which is encoded by a non-rearranging
locus. Pre-TCR-α pairs with the TCR β-chain, which is
the product of a set of somatic DNA rearrangements
that require expression of RECOMBINATION-ACTIVATING GENE 1
(RAG1) (REF. 45) and RAG2 (REF. 46) proteins. At the cell
Unique signal Unique signal surface, the pre-TCR-αβ pair is associated with a collec-
for CD8 lineage for CD4 lineage tion of proteins (the CD3/ζ complex) that is involved in
proximal signal transduction47. Active signalling is
required for further T-cell maturation (TCR β-chain
selection), because mutations that prevent the expres-
sion of key enzymes or adaptors that mediate the gener-
ation of intracellular second messengers by the pre-TCR
complex cause maturational arrest at this DN3
stage48–50. This is the same stage of developmental arrest
that is seen with defective Rag gene expression, which
prevents β-locus rearrangement and hence, pre-TCR
assembly, expression and signalling.
T cells that emerge from β-selection (late DN3 and
TCR–CD8/MHC TCR/MHC class I DN4) undergo 6–8 cell divisions, after which recombi-
class I interaction interaction without
CD8 involvement
nation at the TCR-α locus produces the second com-
ponent chain of the mature αβ antigen receptor. The
expression of pre-TCR-α is lost during this stage,
which results in the cell-surface display of a low level of
MHC- MHC- αβ TCR assembled with CD3/ζ proteins. The thymo-
class-I- class-II- cytes also begin to express co-receptor proteins —
specific specific
TCR TCR most often CD8 first, followed by CD4 (REF. 6) — and,
eventually, form a large population of double-positive
CD8+ CD4+ (DP; CD4+CD8+) αβ-TCR-expressing immature cells
SP SP CD8+
SP that constitute 90% of the lymphoid compartment in
the thymus of young individuals.
Cell survival Cell death
The opposite would occur with a DP cell TCR-mediated selection of DP thymocytes. From this
that expresses a TCR that is specific for
MHC class II large number of DP thymocytes, the subset that is best
suited to function in the host environment is permitted
Figure 2 | ‘Instruction’ and ‘stochastic’ models for the development of mature T cells
to mature and migrate to peripheral lymphoid tissues.
with coordinated TCR and co-receptor specificities. a | The ‘instruction’ model proposes
that biochemically distinct intracellular signals are generated when a T-cell receptor (TCR) and Four distinguishable processes characterize this selec-
CD8 molecule co-engage an MHC class-I ligand compared to when a TCR and CD4 co- tion — death by neglect, NEGATIVE SELECTION, POSITIVE
6,51
receptor co-recognize an MHC class-II ligand. These different messages induce commitment SELECTION and lineage-specific development . Most
to the correct lineage (the CD8 or CD4 pathway, respectively)11,12. Among the genetic events (~90%) DP thymocytes express TCRs that interact so
that are involved in lineage-specific differentiation, the most obvious is loss of expression of the poorly with the available self-peptide–MHC ligands
co-receptor (CD4 or CD8, respectively) that was not involved in generating the signal that
that the intracellular signals that are required to sustain
induced lineage commitment. b | By contrast, the ‘selection’ model suggests that precursor
CD4+CD8+ thymocytes are either already (randomly) committed to a lineage, or, at the start of
viability are not generated, which leads to death
positive selection, make a stochastic lineage choice that is unrelated to the specificity of their by neglect. It is not surprising that so few TCRs
TCR. This results in some cells choosing incorrectly — that is, committing to a developmental bind adequately to self-ligands. The diverse set of
pathway that will extinguish expression of the co-receptor that is required for optimal mature- peptide–MHC-class-I and -class-II structures on corti-
cell antigen responses with the TCR of that T cell. To eliminate such undesired cells, a filter is cal epithelial cells that are involved in initiating T-cell
applied during later development. After one co-receptor is lost, continued differentiation is selection is but a small fraction of the total number of
postulated to be dependent on additional TCR signals that require cooperation with the correct
(MHC-class-specificity-matched) co-receptor. Cells that retain expression of the wrong co-
potential complexes. Although the unselected repertoire
receptor fail to produce this life-sustaining signal and die, so that successfully maturing cells of TCRs is biased towards the recognition of MHC in
have the expected correspondence between TCR and co-receptor specificity for MHC ligands. general52,53, it is unlikely that the random DNA
DP, double positive; SP, single positive. rearrangement and protein-chain pairing process that

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controls the fine specificity of TCRs54 would generate
many receptors that are well-enough matched to the
particular peptide–MHC ligands that are present in any
individual to result in efficient signalling. A small frac-
tion of immature T cells (~5%) bears TCRs that bind
very well to self-ligands; these lymphocytes could cause
autoimmune pathology if they were permitted to leave
the thymus. Signalling on engagement of these TCRs
with self-peptide–MHC ligands promotes rapid apop-
totic death (negative selection). Cells that express TCRs
that recognize self-ligands and generate signals that
have an intensity between those resulting in neglect or
negative selection initiate a multi-step process known
as positive selection that ultimately results in lineage-
specific differentiation into either CD4+ or CD8+
mature T cells.
TCR–co-receptor coordination
The generation of intracellular messengers after TCR
recognition of self-ligands has a crucial role in the pro-
duction of the mature T-cell pool. This involves not
only life or death decisions, but also some mechanism
that permits a differentiating DP thymocyte to coordi-
nate its continued expression of CD4 or CD8 with the
unpredictable specificity of the clonally expressed TCR.
But, why is such coordination important? The answer
lies in the need for sensitivity of peripheral T cells to
foreign antigens.
Antigen-receptor signalling begins with ligand
recognition, which leads to the activation of a SRC-
family kinase, typically lymphocyte-specific protein-
tyrosine kinase (LCK). This kinase is associated with
the TCR–CD3/ζ complex, as well as with the cytoplas-
mic tails of CD4 and CD8 (REF. 55). Activated LCK
tyrosine phosphorylates immunoreceptor tyrosine-
based activation motifs (ITAMs) in CD3/ζ, which
produces a structure that is suitable for tight binding
by the paired SRC-homology 2 (SH2) domains of the
SYK-family kinase ZAP70 (ζ-chain-associated protein
kinase, 70 kDa)56,57. Recruited ZAP70 is phosphory-
lated and activated by the already functional LCK.
Active ZAP70, in turn, tyrosine phosphorylates a
major adaptor protein — linker for activation of
T cells (LAT)58 — as well as several other enzymes and
adaptors. This leads to an increase in the concentration
of intracellular Ca2+, which promotes the nuclear
translocation of nuclear factor of activated T cells
(NFAT) transcription factors and the activation of one
or more mitogen-activated protein kinase (MAPK)
pathways. Together, these events help direct the gene
expression that is necessary for the function of mature
T cells59.
The consequences of TCR interaction with pep-
tide–MHC ligands depend strongly on the ‘quality’ of
the interaction. In particular, suboptimal binding does
not simply result in a smaller quantity of the same sig-
nals, but in a different quality of proximal signalling 60,61.
The capacity of a TCR–ligand pair to interact effec-
tively with either the CD4 or CD8 co-receptor has a
crucial role in producing high-quality signals62,63. The
association of an engaged TCR with a co-receptor
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might facilitate optimal signalling, both through the
action of the additional LCK that is recruited by this
event and the association of co-receptors with the
ZAP70-substrate LAT64. The latter might promote
rapid elevation of intracellular MAPK1 (extracellular
signal-regulated kinase; ERK) activity, which helps to
sustain the function of LCK within the TCR complex60.
As a result, highly effective signalling in response to for-
eign antigen will depend on the coordinate binding of
ligand by the TCR and a CD4 or CD8 molecule.
Therefore, the correct class of co-receptor must be pre-
sent on the mature T cell if it is to be sensitive to low
levels of foreign antigen.
Instruction versus selection
If optimal T-cell function depends on matching the
MHC-class specificity of co-receptor and TCR, how is
coordination between germ-line and somatic specifici-
ties achieved? Thymocyte differentiation involves a
change from the DP to the SP (CD4+CD8− or CD4−CD8+)
state by the silencing of transcription of one co-receptor
locus65–75, which is accompanied by other genetic
events that determine the effector potential of the
mature T cell (‘helper’ versus ‘cytotoxic’ cell). This
polarization could occur randomly due to stochastic
processes before or at the initiation of positive selec-
tion. Alternatively, it could be regulated by the nature
of the initial receptor signal, so that the extinguished
co-receptor locus is (most often) the one with an
MHC specificity that does not match that of the TCR.
In the stochastic model, the remaining co-receptor
would frequently be inappropriate for the TCR of that
cell. A later test of the MHC-class specificity of the
clonal receptor and co-receptor molecule would be
required, so that only cells with the correct match are
allowed to mature. This process of random choice fol-
lowed by testing is known as the ‘stochastic’ or ‘selec-
tion’ model, to distinguish it from the case in which a
specific, unique biochemical event that arises from
initial engagement of the TCR and one of the co-
receptors signals to the T cell to enter the proper lin-
eage (‘instruction’ model)11,12. FIGURE 2 illustrates these
two models.
Rescue experiments support instruction. A series of
experiments in transgenic and gene-targeted (knock-
out) mice was the first to test the competing hypothe-
ses. The selection model predicts that some developing
T cells die because they make the wrong stochastic
choice of which co-receptor to maintain in relation-
ship to their TCR specificity. Therefore, it would be
expected that enforcing expression of the appropriate
co-receptor, in addition to the randomly chosen co-
receptor, should rescue these cells at the developmental
checkpoint that evaluates TCR–co-receptor matching.
The result would be mature T cells that express both
the inappropriate endogenous co-receptor and
the appropriate transgenic co-receptor (mature
CD4+CD8+ T cells). Initial experiments with transgenic
mice that expressed either CD4 or CD8 throughout
T-cell development gave negative results — very few or
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Lineage programme expression Transitional-cell studies argue for selection. The pen-
dulum then swung almost completely towards selec-
tion when several groups reported detailed phenotypic
CD4+CD8low analyses of thymocyte development in mice that were
TCRint/hi deficient in MHC class-I or class-II expression. Guidos
CD4+ SP and colleagues79 had described the transition of DP
TCRhi DP T cells to SP T cells in normal animals as comprising a
TCRlow
series of intermediate states in which the surface
expression of CD4 (on the path to SP CD8+ T cells) or
CD8 (on the path to SP CD4+ T cells) was gradually
CD4

CD4low lost. When thymocytes from MHC class-I-deficient

CD8low/+ CD4lowCD8+ mice were examined, they included substantial num-
TCRint TCRhi
bers of CD4mediumCD8hi cells, a transitional state that
would not be expected to exist if MHC class I mole-
cules were unavailable to ‘instruct’ development along
the CD8 pathway15,16. Likewise, CD4hiCD8medium T cells
were observed in MHC class-II-deficient mice, even
CD8+ SP
TCRhi though the DP T cells could not have been instructed to
DN
develop along the CD4 lineage in such animals15,80. On
the basis of these data, stochastic choice and selection
were invoked to explain TCR–co-receptor matching,
and various arguments were offered to explain the lim-
CD8 ited ability of co-receptor transgenes to rescue the devel-
opment of cells that were predicted to have chosen the
Uncommited CD4-commited CD8-commited wrong expression pattern.
A third model of development was proposed at this
Figure 3 | Complex pattern of CD4 and CD8 expression on positively selected time, known as the default model. Singer and col-
thymocytes. The discovery that CD4+ and CD8+ T cells are derived Nature Reviews
from | Immunology
CD4+CD8 +
double- leagues developed an assay (PRONASE STRIPPING) that
positive (DP) precursors led to the assumption that mature cells that express only one co- allowed analysis of the ongoing, rather than the past,
receptor arise from transcriptional silencing of the irrelevant co-receptor locus and gradual loss state of co-receptor synthesis by an individual T cell.
of surface expression of the corresponding protein. This widely accepted concept of progressive Using this method, they concluded that DP T cells
co-receptor loss contributed to the confusion concerning whether lineage commitment resulted
moved along the CD4 lineage pathway by ‘default’,
from instruction or stochastic choice. Because of the inconsistencies between experiments that
tested predictions of a stochastic/selection model12–14 and unanticipated findings on the lineage
even without TCR engagement, but were deviated
fate of isolated thymocyte subpopulations82, this basic assumption of linear co-receptor from this fate by engagement of the TCR with MHC
extinction was re-evaluated83. These studies revealed that DP cells become CD4+ or CD8+ class I (REF. 81).
single-positive (SP) cells by a complex, non-intuitive route. TCR signalling involving MHC class-I
or class-II recognition results in the partial loss of both CD4 and CD8 expression. These Back to instruction. The divergent conclusions that
CD4lowCD8low cells then begin to re-express CD4, followed by CD8, yielding CD4highCD8medium arose from rescue experiments, analysis of transitional
cells, regardless of whether the initiating signal involves MHC class I or MHC class II ligands. The
extent of CD4 and CD8 downregulation correlates generally with the strength of the initial TCR
cells in MHC-deficient animals and pronase-stripping
signal. So, for cells at the lower limit of signalling that is required for CD8 lineage commitment, studies hinted that some systematic error(s) were pre-
CD8 loss might not be great enough to generate cells with a CD4+CD8low phenotype that are venting the correct interpretation of experiments that
distinguishable from the broad range of co-receptor expression on DP cells. This potentially examined lineage commitment. Several studies helped
explains some reports that CD8-lineage development does not always include passage through to straighten matters out. Two groups showed that
this particular intermediate stage. CD4+CD8low cells that are committed to the CD4 lineage then CD4hiCD8medium T cells could develop into both CD4+
lose expression of CD8 to become CD4+ SP cells. Cells that are committed to the CD8 pathway
and CD8+ SP T cells, in contrast to the prediction of
lose expression of CD4 while gaining expression of CD8; they become CD4+CD8+ again, but
with higher levels of TCR expression than immature DP cells. Eventually, expression of all CD4 is the simple transitional-cell model81,82. Using both
lost and the cells emerge as CD8+ SP cells. This scheme has been verified using molecular kinetic and pronase-stripping analyses, Lucas and
genetic approaches124. Germain then found that this result, and much of the
confusing lineage data, could be explained by the previ-
ous acceptance of an incorrect model of thymocyte
no mature DP T cells were generated12,13. Because the phenotypic change after the DP stage83. It had been
expectation of the selection model was not met, these assumed that DP T cells simply extinguish transcrip-
data were taken as strong evidence for ‘instruction’. tion of the genes that encode CD4 or CD8 and that
Follow-up studies clouded this picture, however. With expression of the corresponding co-receptor disap-
PRONASE STRIPPING
sufficiently high levels of expression of the transgenic pears gradually. But, in reality, co-receptor extinction
The treatment of cells with a co-receptor or with different transgenic TCRs, signifi- occurs in a more complex manner (FIG. 3). TCR sig-
powerful protease that removes cant ‘rescue’ was observed14,76–78. The process was never nalling first leads to some loss of both co-receptors
surface protein molecules, as successful as would be predicted from a simple from the cell membrane, irrespective of whether MHC
followed by the analysis of
surface phenotype immediately
selection model, but the fact that a substantial number class-I or class-II ligands are recognized. The extent of
after such treatment and then of mature DP T cells was seen argued against a this loss seems to correlate with the strength of TCR
after further culture. ‘unique-signal’ instruction model. signalling. Therefore, weaker signals might not cause

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TCR–CD8/MHC class I interaction TCR–CD4/MHC class II interaction enough loss of co-receptor expression to easily distin-
guish these cells from the large pool of ‘neglected’ DP
T cells in the thymus, which explains some reports that
reached a different conclusion about the CD8 pathway
MHC class I MHC class II
of T-cell development (that some cells developing
CD8 CD4
along the CD8 pathway do not undergo this loss of
expression and asymmetric recovery of co-receptor
expression)84,85. Re-expression of CD4 and, with a
delay, of CD8 then occurs. This kinetic asymmetry in
re-expression results temporarily in CD4hiCD8medium
Rare Frequent Rare cells — regardless of whether they have TCRs that are
Long/moderate Short/low Long/moderate Short/low specific for MHC class I or class II — which explains
intensity signal intensity signal intensity signal intensity signal the cell-transfer data from REFS 81,82 and the pronase-
stripping results81. This stage is followed by the loss of
expression of CD4 on cells of the CD8 lineage —
which requires these cells to pass through a DP
CD4mediumCD8medium stage as they become CD8+ SP — or
the loss of expression of CD8 — which allows the cells to
move rapidly and directly from the CD4hiCD8medium state
CD4 committed CD8 committed CD4 committed CD8 committed to the CD4+ SP state.
Once this new scheme of thymocyte development
became clear, the strongest evidence for a
stochastic/selection model disappeared, because the
‘inappropriate’ transitional forms that were seen in
MHC-deficient mice no longer violated the precepts of
an instruction model. The CD4mediumCD8hi cells that
have been observed in MHC class-I-deficient mice can
be explained by the fact that on recognition of MHC
class II, the expression of CD4 is reduced somewhat
more than CD8 during the initial downregulation of
both co-receptors. Logic demanded that if CD4+CD8+
T cells become CD4+ or CD8+ SP T cells, then the DP
cells must lose expression of either CD4 or CD8. This
was a well-founded presumption. However, the extrap-
olation from this that co-receptor loss must occur in a
straightforward ‘linear’ manner was not, as it turned
Inadequate Persistent Inadequate out, correct.
signalling signalling signalling
Molecular mechanisms of lineage choice
‘Strength of signal’ model of lineage commitment. As
CD8+ CD4+ the problems with the transitional-cell analyses became
SP SP
clear, additional studies focused more on the possible
Cell death Cell death molecular mechanisms of ‘instructed’ lineage choice.
Three lines of evidence indicated that the extent of
Figure 4 | ‘Strength of signal’ model for the development of mature T cells with TCR–co-receptor-dependent intracellular signalling
coordinated TCR and co-receptor specificities. The ‘strength of signal’ model proposes had a decisive role in determining the fate of DP T cells
that commitment to the CD4 versus CD8 lineage depends on the intensity23,24,32,36 or
(FIG. 4). First, the cytoplasmic domains of the CD4 and
duration24,33,34,97 of signalling from the T-cell receptor (TCR). Short-duration signals lead to the
CD8 pathway; more prolonged signals lead to the CD4 pathway. Because CD4 is more CD8 co-receptors were exchanged by molecular
extensively associated with lymphocyte-specific protein-tyrosine kinase (LCK) in CD4+CD8+ cloning methods, and these recombinant proteins were
thymocytes30,32,88,89, MHC class-II binding by the TCR and CD4 will tend to produce a expressed in developing T cells22,86,87. The results of
prolonged signal more often than will TCR–CD8 co-recognition of an MHC class-I ligand, these experiments indicated that lineage fate was dic-
resulting in the ‘correct’ lineage choice by most immature T cells. Some of these cells will tated largely by the signalling end of the co-receptor,
express TCRs that only very weakly bind peptide–MHC-class-II ligands, which leads to short
rather than by the class of MHC that was bound. On
signals and an inappropriate CD8-lineage choice. Other cells will have TCRs that bind MHC
class-I ligands particularly well, producing long signals and an improper CD4-lineage choice.
the basis of these data, Itano et al.22 first proposed that
The latter cells will often be eliminated by means of negative selection. The former cells will ‘strength of signal’ dictated the CD4–CD8 decision.
often fail to complete maturation, because the signalling capacity of the TCR without CD4 is Second, Matechek et al.23 found that thymocytes with
inadequate to support subsequent development. This is the same late filtering step that is an MHC class-II-specific TCR became CD8+ SP T cells
postulated in the original ‘selection’ model. Enforced CD4 expression can rescue some MHC when CD4 was absent, rather than failing to mature at
class-II-recognizing T cells that incorrectly enter the CD8 lineage. This can explain the data of all. Given the idea that CD4 assists the TCR in sig-
Leung et al.75 on the effect of a CD4 silencer deletion that maintains CD4 expression in CD8-
lineage-committed cells, without the need to conclude from these findings that the lineage-
nalling when an MHC class-II ligand is recognized,
commitment process is stochastic. SP, single positive. these investigators suggested that weak signalling when

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Box 1 | Analysis of lineage commitment and progression using a two-stage culture model
Jenkinson and Owen first introduced the use of re-aggregate cultures for the analysis of T-cell development134. They
appropriately emphasized the use of this method for the dissection of the role of different cell populations in this process.
In an adaptation of their pioneering method, Yasutomo et al.33,94 separated delivery of the signal that initiates positive
selection of double-positive (DP) T cells from the signals that are required for the completion of maturation to the
single-positive (SP) state. This was accomplished using DP T cells that were isolated from T-cell receptor (TCR)-
transgenic mice that had been bred onto an MHC background that was incapable of initiating either positive or negative
selection, which provided a pool of ‘virgin’ DP T cells. These DP cells were exposed to various peptide–MHC ligands in a
dispersed culture, and then cells with evidence of activation (surface expression of CD69) were isolated by flow
cytometry. Previous studies had shown that thymocytes that are undergoing selection in a normal thymus express CD69
(REF. 135). These activated cells, which have the CD4lowCD8lowTCRintermediate phenotype of early post-selection
thymocytes83,136, were then combined with isolated MHC-deficient thymic stromal cells and additional haematopoietic
dendritic cells of the desired MHC type to provide ligands for the completion of maturation. Previous work, as well as
other studies in this two-step model, had shown that successful maturation depended on such additional TCR
signalling90–94. By varying the quality of the peptide–MHC ligand that is available to the DP T cells in the first culture and
to the activated thymocytes in the second culture, it was possible to determine the stage at which lineage commitment
occurred and the rules that govern this process with respect to TCR and co-receptor engagement. It was also possible to
vary the amount and duration of exposure to the first signal, to investigate how ‘strength of signal’ might influence
lineage choice and/or progression.

DP TCR-transgenic Antigen-presenting Isolate

thymocytes from cells +/– specific CD69hi
non-selecting host peptide cells

Analyse by FACS
+
or function

CD69

First step = overnight Second step = 3 day

dispersed coculture; re-aggregate culture
next day, FACS sort with thymic stroma +/–
for activated CD69+ cells haematopoietic antigen-
presenting cells

CD4 was missing led to the CD8 fate. Third, Iwata and
associates showed that by varying the concentration
and duration of exposure of pre-selection DP T cells to
phorbol ester and ionomycin — which simulate down-
stream signalling by the TCR complex — one could
selectively produce CD4+ or CD8+ SP T cells24. Higher
concentrations and longer exposures produced CD4+
SP T cells, whereas brief exposures to lower concentra-
tions produced CD8+ SP T cells. A similar quantitative
model was proposed on the basis of the effect of the
level of Mapk activity on the generation of CD4+ versus
CD8+ T cells25. Fourth, Zamoyska and associates
showed in thymic-organ cultures that crosslinking the
TCR with either CD4 or CD8, using hybrid antibodies,
led to the emergence of CD4+ SP T cells, whereas
TCR–TCR crosslinking alone produced CD8+ SP
T cells26,29,30. The extent of Lck activation was proposed
to control cell fate31; TCR–co-receptor crosslinking pro-
duced CD4+ SP T cells because this provided more Lck
input than TCR crosslinking alone. The idea, from all of
these studies, that signal strength ‘instructed’ lineage
choice was also supported by evidence that showed that
by manipulating the parameters that affect TCR–co-
receptor signalling, conditions can be achieved in which
almost 90% of DP precursors transit to the SP stage.
This high efficiency would not be expected if lineage
commitment is determined stochastically and an
incorrect choice ‘dooms’ a cell35.
The generation of CD4+ T cells by co-crosslinking
of TCR and CD8 was problematic, however; this result
seemed to be in clear violation of an instructional
model, in which the class of MHC that is co-engaged
by TCR and co-receptor dictates cell fate when physio-
logical ligands, and not antibodies, are involved. Data
on the association of Lck with CD4 and CD8 in imma-
ture thymocytes provided a possible solution. Veillette
et al.88 first reported, and Wiest et al.89 later confirmed,
that CD4 molecules that are isolated from DP cells are
associated with more Lck than are CD8 molecules.
Therefore, under physiological conditions, the co-
engagement of CD4 and TCR by MHC class-II ligands
might promote greater LCK inclusion in the signalling
complex than TCR–CD8 co-recognition of an MHC
class-I ligand. Signalling would, therefore, generally be
much stronger with MHC class-II ligands than with
MHC class-I ligands, providing a screen that ‘instructs’
most DP T cells into the correct lineage upon self-
ligand recognition. The finding that CD4+ SP T cells
were generated by TCR–CD8 crosslinking could then be
explained by postulating that the antibodies markedly

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© 2002 Nature Publishing Group
REVIEWS
exceeded the usual physiological levels of engagement,
and yielded signals that reached the threshold required
for CD4 lineage commitment, even with the lower level
of LCK that is associated with CD8.
Further refinement of the ‘strength of signal’ lin-
eage-commitment model then came from experiments
that involved a two-step thymic-organ culture system33
(BOX 1). DP thymocytes from TCR-transgenic mice that
lacked the MHC ligands required for positive selection
were used as a source of ‘virgin’ precursors. These
immature cells were exposed to MHC ligands for their
TCRs in a first culture step to initiate positive selection,
then recovered and re-aggregated with a mixture of
stromal cells, after which the development of SP T cells
was analysed. The amount and quality of the MHC
ligands in each culture step could be independently
manipulated, which permitted an analysis of how
TCR and co-receptor engagement at each stage affected
lineage-specific differentiation. Recognition of MHC by
the TCR was found to be necessary in both cultures,
which confirmed earlier studies that indicated a need for
persistent receptor signalling in T-cell development90–94.
More importantly, the use of mutant MHC class II mol-
ecules that were unable to interact with CD4 but were
still recognized by the TCR95 revealed that CD4 lineage
fate is instructed by the initial signalling event that is
involved in positive selection, based on whether the CD4
co-receptor is or is not productively co-engaged with the
TCR at that time.
What about CD8+ T-cell differentiation? Given the
TCR–CD8 crosslinking data and these new findings, it
was proposed that very efficient co-engagement of CD8
with a strong class-I-binding TCR could produce suffi-
cient signal to direct DP T cells into the CD4 lineage.
Such fate choices would normally be undetectable,
because cells that expressed a TCR with such high affin-
ity for a self-MHC ligand would undergo negative
selection (apoptosis) before completing maturation.
However, in the two-stage culture system, such late
deletion could be avoided by replacing the potent lig-
and with a less avid one that still supported receptor
signalling in the re-aggregate phase. This prediction
was tested using HY-specific-TCR-transgenic T cells.
Cells that carry this TCR usually die in the presence of
the strongly binding, MHC class-I-associated male HY
antigen96, but develop into CD8+ SP T cells in the pres-
ence of female cells that lack this antigen8. Remarkably,
when first exposed to male cells and then re-aggregated
with female cells, HY-specific-TCR-expressing precur-
sors developed into SP CD4+ T cells, rather than CD8+
T cells33 — similar to the results of Bommhardt et al.26
using TCR–CD8 crosslinking with antibodies.
Watanabe et al. reported similar results that showed
that varying the magnitude of stimulation of thymo-
cytes with a transgenic TCR that is specific for an MHC
class-I ligand resulted in the generation of CD8+ SP
T cells at low ligand levels and CD4+ SP T cells at
increased concentrations of antigen36.
What does ‘strength of signal’ mean? In the re-
aggregate system described, limiting the quantity of lig-
and that was available to the TCR without influencing
316 | MAY 2002 | VOLUME 2
© 2002 Nature Publishing Group
co-receptor function failed to alter cell fate33. On the basis
of, in part, biochemical data24,29, signal duration was con-
sidered as an alternative candidate to ligand concentra-
tion. This hypothesis was tested by limiting the length of
exposure of HY-specific-TCR-transgenic DP T cells to
male antigen in the first culture. When the interaction
was limited to a few hours, the cells became CD8+ SP
T cells in the second culture, whereas if the DP cells were
exposed to HY antigen for more than 14 hours, they
developed into CD4+ SP T cells. These data tied together
the preceding observations that concerned signal
strength in lineage choice by indicating that co-receptor
binding contributes to the maintenance of signalling
above some threshold level, and that cell fate is respon-
sive to the length of this supra-threshold input.
Experiments in mature T cells have shown that the cou-
pling of Lck to the co-receptor helps to dictate the quality
of receptor signalling62, which, in turn, affects the rate of
feedback desensitization and hence, the duration of sig-
nalling61. This hypothesis is also consistent with the
effects of altered Lck activity or amount on the CD4–
CD8 choice31,32 and with data on the role of the duration
of protein kinase C (Pkc) and Mapk1 activation in CD4
versus CD8 lineage commitment24,97.
However, pronase treatment of cells that were recov-
ered from the first-step cultures in the re-aggregate
model showed that both co-receptor loci were still
active, even though lineage fate was already fixed. This
meant that it was possible to separate commitment to
the CD4 versus CD8 lineages from progression along
these differentiation pathways, at least in terms of silenc-
ing of the relevant co-receptor loci. Differentiation
apparently requires additional signals that are, perhaps,
dependent on epithelial elements in the thymus. The
evidence from these experiments that thymocytes are
fully lineage-committed after a few hours of TCR sig-
nalling but don’t have lineage-specific co-receptor
silencing, as well as data that show that CD8-lineage
commitment precedes CD4-lineage commitment tem-
porally97,98, is inconsistent with a distinct ‘duration of
signal’ model of development, in which lineage choice is
proposed to default to the CD4 pathway on initiation of
TCR signalling and to be reversed to CD8-lineage devel-
opment in the case of T cells that recognize an MHC
class-I ligand if TCR signalling is abrogated within a few
hours due to loss of CD8 expression34 (FIG. 4).
Imperfections in the lineage commitment process. A
fate-commitment mechanism that is based on the
quantitative parameter of signal duration cannot be
perfect. The initial DP T-cell pool will contain TCRs of
MHC class-I and class-II specificity that have overlap-
ping ranges of affinities for their respective ligands. In
the context of the commitment model described, this
must inevitably lead to ‘wrong’ choices, even with
asymmetric co-receptor–LCK association. Some cells
with TCRs that bind weakly to MHC class-II ligands
will begin developing along the CD8 pathway and some
that have strong MHC class-I binding will opt for the
CD4 fate. Such ‘incorrect’ lineage choice presumably
accounts for the ‘rescue’ of some developing T cells in
www.nature.com/reviews/immunol
 REVIEWS

P For this reason, mismatches between the class of

MHC that is involved in TCR stimulation and
SHP1 CD4–CD8 fate choice, as revealed in the gene-targeting
studies, do not necessarily argue against instruction and
support stochastic commitment, as some investigators
– have proposed75,100 (see legend to FIG. 2 for details).
MHC SHP1 Rather, these mismatches are merely inconsistent with
an instruction model that postulates the control of lin-
TCR
P Y394 P Y394 eage choice by qualitatively unique signals that arise
from CD4 versus CD8 co-engagement. The existence of
S59
p56 LCK p56 LCK p59 LCK
P
‘mismatched’ cells is fully in accord with the predicted
inability of a pathway that combines quantitative
instruction with post-commitment selection to ensure
+ an absolute match between TCR specificity and cell fate.
It seems that some inaccuracy in the system of cell-fate
Relative generation rates
P
choice among developing T cells is not sufficiently dam-
Antagonist peptide aging for evolutionary pressures to have selected against
MAPK1 MAPK1 P this occurrence.
P P

SHP1 MAPK1
Biochemistry and cell biology of lineage choice. How
Agonist peptide
do variations in the strength and/or duration of
TCR–co-receptor signalling dictate lineage choice?
P P
Unfortunately, the information that is available on
MAPK1 SHP1 this important issue is limited at present. In part, this
is because many of the relevant molecules also have
Figure 5 | Two different roles for MAPK1 in thymocyte development. A large body of literature roles in earlier stages of thymocyte differentiation. For
supports a key role for mitogen-activated protein kinase 1 (MAPK1) in thymocyte selection and example, genetic inactivation of the Zap70 (REF. 48),
lineage-specific differentiation of thymocytes. Active MAPK1 seems to be crucial to successful Lck and Fyn49, or Slp76 (SH2-domain-containing
positive selection in general125–130, and higher levels of MAPK1 activity promote thymocytes to protein of 76 kDa)50 loci results in the arrest of devel-
become CD4+ single-positive (SP) T cells, whereas interference with MAPK1 activity results in opment at the DN3 stage (β-checkpoint), before the
excess CD8+ SP T-cell generation25,101. This kinase was, until recently, presumed to mediate its
CD4–CD8 decision.
effect on these developmental processes through its action on nuclear transcription factors, and
there is strong evidence that this is the case. For example, MAPK1 has been implicated in
Despite these limitations, some informative experi-
induction of the transcription factor early growth response 1 (EGR1) and the subsequent ments exist. Expression of a dominant active Mek1
expression of inhibitor of DNA binding 3 (ID3), which is necessary to inactivate E2A and permit (MAPK kinase 1) transgene — which activates Mapk1
T-cell development beyond the double-negative (DN)1 stage131,132. But, two other sets of — promotes the development of CD4+ T cells at the
experiments raise another, non-mutually exclusive, possibility. First, the data from the two-stage expense of CD8+ T cells25. Conversely, blockade of the
culture model indicate that the duration of T-cell receptor (TCR)–co-receptor signalling controls Mapk1 pathway results in a reduction in the generation
lineage choice33, and the work of Wilkinson and Kaye implicates the persistence of MAPK1
activation as a key aspect of the duration-controlled lineage decision97. Second, as shown in the
of CD4+ T cells and an increase in CD8+ T-cell produc-
figure, MAPK1 has been found to be the central element in a positive-feedback loop that regulates tion25,101. In a tumour-cell model, active RAS promotes
the duration of TCR signalling in mature T cells60. Active MAPK1 phosphorylates serine at position the loss of CD8 expression102, which is consistent with
59 and activates lymphocyte-specific protein-tyrosine kinase (LCK). This phosphorylation limits the data that indicate a role for the MAPK pathway in lin-
ability of SRC-homology-2-domain-containing protein tyrosine phosphatase 1 (SHP1) to bind and eage choice. The same laboratory has shown very
dephosphorylate LCK (crossed arrow). When SHP1 is recruited to the TCR complex, it inactivates recently that the duration of activation of MAPK1
LCK and ZAP70 (ζ-chain associated protein kinase, 70 kDa), which terminates T-cell
seems to have a key role in CD4+ versus CD8+ T-cell
signalling103–110. So, MAPK1 activity leads to prolonged TCR signalling by preventing SHP1 from
limiting the duration of LCK and ZAP70 kinase function. This would favour the entry of cells into the development97,98; more than ten hours of elevated
CD4 pathway, whereas blockade of MAPK1 activation would result in truncated signalling that MAPK1 activity is necessary for cells to become CD4+
promotes a CD8-lineage decision — as seen in experiments that modify MAPK1 activity in these SP T cells, which agrees with the data from the two-
two directions. Furthermore, MAPK1 is selectively involved in this feedback loop and is non- stage culture experiments33. Interestingly, a short
redundant for efficient T-cell development133. So, in addition to any direct effect of MAPK1 on the period of MAPK1 activity is crucial for the appearance
transcriptional control of gene expression, MAPK1 might contribute to the control of thymocyte
of CD8+ SP T cells in this model, because such differen-
fate indirectly through its control of TCR signalling, which could then act through other downstream
mediators to regulate the differentiation process. Distinguishing between the roles of the TCR
tiation is prevented by the complete blockade of the
feedback and direct downstream effects of MAPK1 on thymocyte development will require the MAPK pathway. It was suggested that CD8-lineage
generation of animals with selective mutations in LCK that affect only the TCR-feedback function development is the default pathway that is selected
of MAPK1 and not its nuclear activities. when initial signalling exceeds the ‘neglect’ threshold.
Prolonged MAPK1 signalling presumably overrides
this default condition and results in the generation of
mice when persistent co-receptor expression is CD4-lineage-committed T cells. In this regard, this
enforced through transgenic14,76–78 or gene-targeting75 model is the polar opposite of the co-receptor reversal
strategies, and the ‘incorrect’ co-receptor expression hypothesis of Singer and associates34. These data on the
that is observed on cells exposed to particular duration of MAPK signalling and lineage-choice do
TCR–MHC combinations99. agree with the findings of Ohaka et al.24.

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REVIEWS

Box 2 | NOTCH and T-cell development

NOTCH is a transmembrane protein that is cleaved during
biosynthesis by a furin-like activity to produce a non-
covalently associated extracellular fragment and a
transmembrane/cytoplasmic fragment. The interaction of
NOTCH with one of its ligands (jagged 1 (JAG1), JAG2 or
NOTCH ligand
δ-like 1 (DLK1) in mammals) induces a further cleavage of the
molecule just inside the plasma membrane, which is mediated
by presenilin/γ-secretase. The released intracellular fragment NOTCH1–4
(NOTCH-IC) regulates gene expression by its effects on
C-promoter-binding factor 1 (CBP)/Su(H) and Deltex.
The former activates the HES (hairy and enhancer of split)
and nuclear factor-κB2 (NF-κB2) family of transcription
factors, whereas the latter inhibits JUN N-terminal kinase NOTCH-IC
(JNK) pathway signalling113. The regulation of NOTCH
function by several co-modifiers, such as Lunatic fringe137, is
not shown. The general model of NOTCH function in cell-fate
specification is that NOTCH signalling leads to the adoption
of a secondary fate, whereas the absence of NOTCH signalling CBF/Su(H) Deltex
allows a cell to adopt a default or primary fate113.
On the basis of data that were obtained using
overexpression of a constitutively active Notch1 transgene,
HES, NF-κB2 and JNK
NOTCH signalling was first proposed to affect thymocyte other mediators pathway
development by promoting the secondary fate of CD8-
lineage development in concert with ongoing TCR
signalling, whereas TCR signalling alone would lead to the
primary fate of CD4-lineage development115. However,
Yasutomo et al. did not find evidence of a role for Notch1 in
the CD4–CD8 decision in their two-step culture model33.
Instead, they observed a role for Notch in the differentiation of already committed cells along the CD8, but not the
CD4, developmental pathway. Deftos et al. concluded that Notch signalling contributed to the upregulation of
expression of B-cell lymphoma protein 2 (Bcl2)117. Animals that were transgenic for an active Notch1 fragment —
similar, but not identical, to that used by Robey et al. — had an accumulation of CD8+ T cells that did not enter the
peripheral pool, which Deftos et al. likened to results obtained by the transgenic expression of Bcl2 in thymocytes138.
No loss of CD4+ T cells was seen in these studies, although more recent analysis by others of younger animals from this
transgenic line indicates that the phenotype is similar to that seen by Robey et al.115. Wolfer et al. eliminated expression
of Notch1 using a conditional gene-targeting strategy, in which the inactivation of the locus occurs only after
commitment to the T-cell lineage118 and hence, does not affect the T-cell–B-cell decision as does germ-line Notch1
deletion39. Under these conditions, these investigators found no effect on CD4+ and CD8+ T-cell generation, from
which it was concluded that Notch1 had no role in later stages of T-cell development. These divergent data cannot be
fully reconciled at present, although the main text suggests some possible explanations. Figure adapted, with
permission, from REF. 111 © Oxford University Press (1999).

It is not known how the MAPK1 pathway influences
lineage fate, because very few target genes have yet to be
defined as being expressed specifically by one or the
other lineage, other than the co-receptor loci. Both co-
receptor genes have very complex sets of promoters
and enhancers; CD4 also uses a specific silencer ele-
ment for regulation65–75. Different regulatory elements
control the initial expression of CD4 and CD8 by
T cells that pass the β-checkpoint; the extinction of
expression of a co-receptor during lineage progression
after the initiation of selection; and the maintenance of
expression of one co-receptor on cells of a given lin-
eage. The biochemical events that link proximal TCR
signals to the activity of these various co-receptor
genetic regulatory elements remain to be defined. In
addition, evidence that MAPK1 has a feedback regula-
tory role in TCR signalling 60 complicates interpretation
of the data on the control of lineage fate by this kinase
(FIG. 5). If active MAPK1 helps to delay receptor desensi-
tization in thymocytes by SH2-domain-containing
protein tyrosine phosphatase 1 (SHP1), as it does in
mature T cells103–110, the manipulations of Mapk1 activ-
ity in these various studies might not produce their
effects on lineage fate only by acting downstream on
gene transcription, as is commonly assumed. Rather,
MAPK1 might contribute to lineage choice by prolong-
ing TCR signalling when LCK-coupled co-receptor
recruitment is adequate, so as to reach the threshold
duration for commitment to the CD4 lineage.
Additional studies that separate the proximal feedback
effects of MAPK1 from the gene-regulatory effects are
needed to clarify this issue.
Notch and CD4–CD8 lineage choice and progression.
Another receptor–ligand system has also been pro-
posed to have a central role in controlling thymocyte

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© 2002 Nature Publishing Group

development (BOX 2). NOTCH and its ligands are
expressed early in thymic development on both T-cell
precursors and stromal elements111–114. Robey and
associates reported that in the presence of TCR–MHC
signalling, a constitutively active Notch1 transgene
promotes the development of CD8+ SP T cells at the
expense of CD4+ SP T cells, and that CD8+ SP T cells
are generated in these transgenic animals even when
only MHC class-II ligands are available to the TCR of
the DP T cell115. Their interpretation of these data was
that in the presence of a permissive TCR signal, Notch
dictates the lineage fate of thymocytes, as it does for so
many other developing tissues. Others have also
reported an effect of Notch on the CD4:CD8 ratio116.
This conclusion has been challenged, however, by sub-
sequent studies. Deftos et al. reported that a similar,
active Notch1 transgene promotes thymocyte sur-
vival117. They concluded that active Notch signalling
results in the accumulation of the same immature
CD8+CD4− thymocytes that are seen in transgenic
mice that express the anti-apoptotic protein B-cell
lymphoma protein 2 (Bcl2), and that Notch does not
affect CD4- versus CD8-lineage choice. This picture is
complicated by a more recent analysis of younger
mice of this strain, which gives a picture that is more
similar to that of Robey and colleagues (B. J. Fowlkes,
personal communication). Using the re-aggregate sys-
tem, Yasutomo et al. also found no evidence for
Notch-mediated control of lineage commitment33.
However, in contrast to Deftos et al., these investiga-
tors did see a selective contribution of Notch to post-
commitment progression along the CD8, but not
CD4, pathway. Finally, any role for Notch1 in thymo-
cyte development after the β-checkpoint has been
called into question by the absence of an effect of
selective Notch1 gene deletion at the DN stage on
CD4+ or CD8+ SP T-cell differentiation118.
At present, it is not possible to propose a fully
coherent model that incorporates all of these data.
One way to reconcile the active Notch1-transgene115
and reaggregate-culture33 data is to draw on the obser-
vations of Pear and co-workers119. These investigators
found that the overexpression of active Notch1 down-
regulated TCR signalling in developing thymocytes
and blocked maturation. Several laboratories have
reported that TCR signalling in response to the type
of weak ligands that promote positive selection
becomes less efficient as a cell progresses past the DP
stage120,121. Therefore, it is possible that the overexpres-
sion of active Notch pushes early T cells along this
pathway of diminished TCR signalling capacity. This
would, in turn, put a larger fraction of the cells into
the low/short signal-duration pool, which promotes
CD8- rather than CD4-lineage development, as
observed by Robey et al. This would not be due to a
direct effect of Notch on lineage choice, but to an
indirect effect on TCR signalling. The findings of
Yasutomo et al. are consistent with this suggestion —
loss of Notch expression did not affect fate choice,
only the development of already committed cells
along the CD8 maturation pathway.
NATURE REVIEWS | IMMUNOLOGY
© 2002 Nature Publishing Group
REVIEWS
This still leaves the data by Radkte et al. on Notch1
deletion to be explained. One can either conclude that
the transgenic and in vitro studies are both unphysio-
logical and irrelevant, or consider the possibility that
members of the Notch family other than Notch1 (par-
ticularly Notch2) might be responsible for the effects
seen by Yasutomo et al. and produce the physiological
counterpart of the signalling that is induced using
active Notch1 transgenes. Immature thymocytes
express several NOTCH-family members111,113,114, so
only additional studies will answer this question. One
reason to continue to focus on NOTCH as a potential
player in post-signalling lineage progression comes
from the identification of sites in the regulatory regions
of CD4 that bind HES1 (hairy and enhancer of split 1)73
— a key signalling molecule in the NOTCH pathway113.
Although evidence is lacking that such binding controls
the expression of CD4 in a manner that is consistent
with its silencing in cells that are developing in the CD8
pathway, this is a tempting possibility.
Conclusion
The past few years have seen substantial progress in
elucidating the molecular genetic basis for commit-
ment to the T-cell versus B-cell lineages and for effec-
tive progression from immature to mature T cells, irre-
spective of co-receptor-defined lineage122. Advances at
this level of resolution are still lacking with respect to
the commitment of precursor T cells to the CD4
(helper) versus CD8 (cytotoxic) pathways. This is not
to say that the field has not moved forward. Although
not all workers interpret the available data in the same
way, the bulk of evidence appears to favour a model in
which the extent/duration of signalling that results
from TCR and co-receptor engagement of self-pep-
tide–MHC ligands controls the lineage choice of bipo-
tential DP αβ T cells. Because the decision is based on
a quantitative effect of ligand recognition, and the
range of TCR recognition affinities on precursor cells
is broad and overlapping between those that preferen-
tially engage self-peptide–MHC-class-I versus -class-II
ligands, errors are made in the initial decision process.
These mistakes are largely, but not completely, cor-
rected during the later stages of maturation, as co-
receptor extinction takes place. So, elements from
both of the two competing hypotheses that concern
thymocyte lineage commitment have been found to
contribute to the developmental process — ‘sloppy’
instruction at the first step and ‘leaky’ selection at the
second step (FIG. 2). The end result is a pool of mature
T cells that, for the most part, possesses the coordina-
tion of TCR and co-receptor specificity that is
required for effective immune responses to foreign
antigens. Whether ‘mistakes’ that survive are simply
developmental ‘noise’ of no consequence, or whether
such cells might have particular roles in autoimmune
disease, remains unknown. With some luck, it will be
only a few years before another review of this topic
can provide a nearly complete map of CD4- and
CD8-lineage development from the cell surface to the
genome and back.
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REVIEWS
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Acknowledgements
I wish to thank the colleagues in my own laboratory and around the
world whose experiments and discussions have helped to shape
© 2002 Nature Publishing Group
the ideas in this review. I also thank B.J. Fowlkes for reading a draft
of this manuscript and making many very helpful suggestions for
corrections, as well as improvements in presentation. Any errors
that remain are my own.
Online links
DATABASES
The following terms in this article are linked online to:
InterPro: http://www.ebi.ac.uk/interpro/
ITAM | SH2 domain
LocusLink: http://.ncbi.nlm.nih.gov/LocusLink/
Bcl2 | CD3/ζ | CD4 | CD8 | CD25 | CD44 | CD69 | Deltex | DLK1
| E2A | EGR1 | Fyn | HES | HES1 | HY antigen | ID3 | JAG1 |
JAG2 | JNK | LAT | LCK | Lck | Lunatic fringe | MAPK1 | Mapk |
Mek1 | MHC class I | MHC class II | NFAT | NF-κB2 | Notch1 |
Notch2 | Pkc | presenilin | Rag | RAG1 | RAG2 | SHP1 | Slp76 |
ZAP70 | Zap70
Access to this interactive links box is free online.
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