1996 Oxford University Press
International Immunology, Vol. 8, No. 5, pp. 773-780
Developmentally regulated expression of the
PD-1 protein on the surface of doublenegative (CD4~CD8~) thymocytes
Hiroyuki Nishimura12, Yasutoshi Agata1, Akemi Kawasaki4, Masaki Sato3,
Sadao Imamura2, Nagahiro Minato3, Hideo Yagita4, Toru Nakano1 and
Tasuku Honjo1
Departments of 1 Medical Chemistry, 2Dermatology and 3lmmunology, Faculty of Medicine,
Kyoto University, Yoshida, Sakyo-ku, Kyoto 606, Japan
department of Immunology, Juntendo University, School of Medicine, Hongo, Bunkyo-ku, Tokyo 113,
Japan
Keywords: anti-CD3 stimulation, double-negative thymocytes, PD-1, RAG 2~/~mice, T cell development
Abstract
PD-1, a member of the Ig superfamily, was previously isolated from an apoptosis-induced T cell
hybridoma 2B4.11 by subtractive hybridization. Expression of the PD-1 mRNA is restricted to
thymus in adult mice. Using an anti-PD-1 mAb (J43), we examined expression of the PD-1 protein
during differentiation of thymocytes in normal adult, fetal and RAG-2"7" mice with or without antiCD3 mAb stimulation. While PD-1 was expressed only on 3-5% of total normal thymocytes, -34%
of the CD4CD8" double-negative (DN) fraction are PD-1 + cells with two distinct expression levels
(low and high). PD-1 high thymocytes belonged to TCR 76 lineage cells. In the DN compartment of
the TCR af) lineage, PD-1 expression started at the low level from the CD44 + CD25 + stage and the
majority of thymocytes expressed PD-1 at the CD44~CD25~ stage in which thymocytes express TCR
P chains. The anti-CD3e antibody administration augmented the PD-1 expression as well as the
differentiation of the CD44~CD25+ DN cells into the CD44CD25" DN stage, not only in normal mice
but also in RAG-2-deficient mice. The fraction of the PD-1 l0W cells in the CD4 + CD8 + doublepositive (DP) compartment was very small (<5%) but increased by stimulation with the anti-CD3
antibody, although the total number of DP cells was drastically reduced. The results show that
PD-1 expression is specifically induced at the stages preceding clonal selection.
Introduction
The adult thymus contains T cell progenitors of the TCR aP
lineage at several distinct maturation stages which are defined
by the expression level of the CD4 and CD8 co-receptors.
The most immature T cell progenitors in thymus are CD4~
CD8~ double-negative (DN) cells (1), which will subsequently
develop into CD4 + CD8 + double-positive (DP) cells. These
DP thymocytes undergo DNA rearrangements in the TCR a
chain locus, and are subsequently subjected to positive and
negative selections (2-4). Only positively selected thymocytes
mature into CD4 + or CD8 + single-positive (SP) cells. Within
the DN compartment, a maturation process has been further
defined by the expression of the IL-2 receptor a chain (CD25)
and the phagocytic glycoprotein 1 (CD44). CD44+CD25" DN
cells mature in the order of CD44 + CD25 + , CD44~CD25+ and
CD44"CD25" cells (5,6). On the other hand, a maturation
process of TCR yd lineage cells has not been well defined
with these surface markers.
Recent studies on mutant mice of the RAG-1, RAG-2 and
TCR genes (7-9) have highlighted the importance of TCR p
expression for ap T cells to differentiate from the DN to DP
stages. Thymocyte development in RAG-1 or RAG-2 mutant
mice is blocked at the CD44"CD25+ DN stage (7,8). The
maturation beyond the CD44"CD25+ DN stage, at which TCR
P chain gene rearrangement takes place (10), would require
signaling through the TCR P chain (11) and pre-Ta (pTa)
heterodimer (12-14). Signaling through the TCR p chain and
pTa heterodimer was substituted by i.p. administration of an
anti-CD3e mAb into RAG-2-deficient mice (15). In this system,
CD25 + DN thymocytes develop synchronously into the DP
stage. Similar synchronous differentiation and expansion of
Correspondence to: T. Honjo
Transmitting editor. M. Miyasaka
Received 20 November 1995, accepted 6 February 1996
774
PD-1 expression in early thymocytes
DN thymocytes have been shown in the ontogeny of normal
mice (16).
Recently, cDNA for a new surface maker PD-1 was isolated
from apoptosis-induced T cell hybridoma (17). The PD-1
mRNA expression is restricted to the thymus in normal mice
and augmented by i.p. administration of the anti-CD3e (1452C11) mAb. PD-1 belongs to the Ig superfamily and contains
the cytoplasmic immunoreceptor tyrosine activation motif
(18), which is thought to be involved in intracellular signal
transduction of the CD3 y8e and C, family proteins. Although
these data suggest that PD-1 may have some roles in T
cell apoptosis and/or development, the function of PD-1
remains elusive.
In this report, we studied regulated expression of the PD1 protein during maturation of DN thymocytes using a mAb
against PD-1 (J43) that was recently raised to characterize
PD-1 expression at protein levels (19). We found that -34%
of CD4"CD8" DN cells express PD-1 in normal mice. We
characterized PD-1 expression on the DN subpopulation
classified by the two early thymocyte markers, CD44 and
CD25, and revealed that the low level of PD-1 expression on
TCR <xp lineage cells is induced at the CD44 + CD25 + DN
stage and is predominant on the majority of CD44~CD25~ DN
cells which express the TCR p chain. PD-1 expression is
ceased at the DP stage. Moreover, anti-CD3 mAb stimulation
augmented PD-1 expression on CD44~CD25~ DN cells in
normal as well as RAG-2~'~ mice.
A) total thymocytes
78
14
5sTo
•>•«
C57BL/6 (B6) and time-pregnant B6 mice were purchased
from Japan SLC (Shizuoka, Japan). RAG-2"'" mice (7) were
provided by F. Alt (Harvard) and maintained in our specific
pathogen-free facility.
Antibodies
J43 mAb (hamster IgG anti-mouse PD-1) (19) and 145-2C11
mAb (hamster IgG anti-CD3e) (20) were used after purification
with Protein A column chromatography. J43 was conjugated
to biotin (19) or FITC (Cappel, Durham, NC). The anti-NK1.1
mAb (clone PK136) (21) was conjugated to biotin. Hamster
IgG was obtained from Cappel. The following mAb used in
this study were purchased from PharMingen (San Diego,
CA): phycoerythrin (PE)-coupled anti-CD4 (clone RM4-5),
PE-coupled anti-CD44 (clone 1M7), FITC-labeled anti-CD25
(clone 7D4), FITC- and biotin-labeled anti-TCR ap (clone
H57-597), biotin-conjugated anti-TCR Vp8.1,8.2 (clone MR52), biotin-conjugated anti-TCR y5 (clone GL3), and PE-conjugated anti-NK1.1 (clone PK136). FITC-labeled anti-CD8a mAb
(clone 53-6.7) and Red670-streptavidin (SA) were obtained
from Gibco/BRL (Gaithersburg, Maryland). PE-SA was purchased from Dako (Glostrup, Denmark). Anti-c-/c/f mAb
(ACK2) conjugated to biotin is a gift from S.-l. Nishikawa
(Kyoto). For the cytotoxic treatment of thymocytes, the culture
supernatants of clone RL172.4 (anti-CD4, rat IgM) and clone
3.155 (anti-CD8a, rat IgM) were used together with complement.
Total
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C) DN thymocytes
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Methods
Mice
-
• PD-1
Fig. 1. PD-1 expression on total and DN thymocytes from C57BL/6
mice. (A) Total thymocytes from C57BL/6 were analyzed by threecolor flow cytometry with anti-CD4, anti-CD8a and anti-PD-1 mAb.
Control background fluorescence was determined in the absence of
the PD-1 mAb (J43). Twenty thousand events were collected. (B)
DN-enriched thymocytes were analyzed by two-color staining with
anti-PD-1 and anti-CD25 mAb. The purity of DN cells was -99.4%.
The bars in the PD-1 histogram of DN thymocytes show two expression
levels of PD-1. (C) DN-enriched thymocytes were analyzed by threecolor staining with anti-CD44, anti-CD25 and anti-PD-1 mAb. The four
panels in bottom right reveal PD-1 expression in the subsets gated
in the CD44-CD25 panel. Numbers in contour profiles indicate the
percentage of each population.
Flow cytometry
Freshly prepared thymocytes from C57BL/6 or RAG-2"'" mice
were suspended in a staining buffer containing PBS, 2% FCS
and 0.2% azide. Cells were first incubated with 2.4G2 (antiFc receptor) to prevent non-specific binding of mAb through
Fc receptors. In general, 5x10 5 cells were incubated on ice
for 20 min with indicated staining reagents, at saturating
concentrations. The cells were washed with 1 ml of the staining
buffer. In the case of two-color staining using biotinylated
antibodies, PE-SA was used as the secondary reagent.
When three-color staining was performed, the cells were
resuspended in 50 til of appropriately diluted Red670-SA
and subjected to another 15 min incubation. The cells were
washed once and resuspended in 500 \i\ of the staining
buffer. Analysis was performed using a FACScan flow cytometer (Becton Dickinson, Mountain view, CA) and Lysys II
PD-1 expression in early thymocytes
Software (Becton Dickinson). A total of 20,000 events were
collected and viable cells were determined by forward and
sideway scatters. The condition of set quadrants was determined using control staining. The contour profiles were shown
with 10% probability.
A) TCR op NK cells
775
NK1.1+CD44+
In vivo anti-CD3 mAb treatment
Between 500 and 800 ng of purified 2C11 mAb were injected
i.p. into C57BL/6 and RAG-2"'" mice, and the thymocytes
were analyzed by flow cytometry at the indicated times after
injection.
CD4~CD8~ thymocyte preparation by complement-mediated
killing
Thymocytes were collected from two to three C57BL/6 mice
at 4-5 weeks of age with or without 2C11 injection. CD4~
CD8~ thymocytes were prepared by incubating thymocytes
with anti-CD4 (RL172.4) mAb, anti-CD8 (3.155) mAb and
Low-Tox-M rabbit complement (Cedarlane, Ontario, Canada)
at 1:10 dilution for 40 min at 37°C. Live cells were washed
twice with PBS and subjected to an additional 1 h of incubation
for killing. The collected cells were examined for the degree
of purity by flow cytometry using the anti-CD4 (RM4-5) and
ant-CD8 (53-6.7) mAb. A purity of 97-99% was usually
obtained.
Results
PD-1 expression on DN cells from normal adult mice
Subsets of thymocytes from adult C57BL/6 mice were analyzed for expression of PD-1 DP, CD4+CD8~ SP, CD4-CD8 +
SP and DN thymocytes contained - 5 , 10, 15 and 38% PD1 + cells respectively (Fig. 1A and data not shown). In order
to confirm PD-1 expression on DN cells, DN thymocytes were
enriched by cytotoxic elimination of the other subsets with
the anti-CD4 mAb, anti-CD8 mAb and complement. PD-1 was
detected on 34% of the enriched DN population, and two
expression levels of PD-1, low (24.7%) and high (9.3%), were
clearly distinguished (Fig. 1B).
We further investigated which subsets in the DN population
expressed PD-1 using the two early thymocyte markers, CD25
and CD44, because the differentiation of DN thymocytes in
the TCR aP lineage is believed to proceed in the order
of CD44+CD25-, CD44 + CD25 + , CD44'CD25 + and CD44"
CD25-(5,6). As shown in Fig. 1(C), 30% of the CD44 + CD25"
DN thymocytes expressed low (13%) or high (17%) levels of
PD-1. Around 13.5% of CD44 + CD25 + and 28.3% of CD44""
CD25 + DN cells expressed the low level of PD-1 but none of
them expressed the high level of PD-1. However, the major
fraction (82%) of CD44~CD25~ cells expressed either low
(27.3%) or high (55%) levels of PD-1. In the profile of CD25/
PD-1, the CD25 + PD-r o M o w population and CD25-PD-1hi9h
population were distinctly separated (Fig. 1B).
PD-1 expression in the TCR aP NK cell and TCR y8 lineages
of normal adult mice
The DN compartment is heterogeneous and contains TCR aP
NK cells and TCR 78 lineage cells in addition to immature TCR
aP lineage cells. To further define the relationship between
CD44-CD2S+
TCH.it -
Fig. 2. PD-1 expression in the TCR a(5 NK and TCR y8 lineage cells.
(A) DN thymocytes were enriched and analyzed with anti-CD44,
anti-NK1.1 and anti-PD-1 mAb. PD-1 expression is shown in the
CD44+NK1.1 + subset gated in the profile of CD44 and NK1.1 staining.
(B) DN thymocytes were analyzed with anti-PD-1 and anti-TCR y8
mAb. TCR y5 thymocytes expressed PD-1 at high levels. The four
right panels revealed TCR y8 expression in the subsets of DN
thymocytes based on the expression of CD25 and CD44.
PD-1 expression and the differentiation of TCR ap lineage
thymocytes, we examined PD-1 expression in other T lineage
cells such as TCR 78 lineage and TCR aP NK cells. The
CD44*CD25~ DN compartment contains a subset of TCR aP
thymocytes expressing both CD44 and NK1.1(22), but not ckit (23) that is indicative of the immature phenotype. The
usage of the TCR p chain in this subset is skewed toward
Vp8 (22,24). As shown in Fig. 2(A), a distinct population
(5.4%) positive for both CD44 and NK1.1 was detected in
the DN compartment. The thymocytes in this population
expressed TCR aP but not c-kit (data not shown). About
50% of these thymocytes used Vp8 (data not shown). This
population is therefore likely to be TCR <xp NK cells, 33% of
which expressed the low level of PD-1 (1.8% in the DN
compartment), but not the high level (Fig. 2A). NK + cells were
contained exclusively in the CD44+CD25~ compartment (data
not shown). Thus the majority of the PD-1 low thymocytes in
the CD44+CD25~compartment (2.0% in the DN compartment)
(Fig. 1C) are likely to be TCR ap NK cells.
By contrast, almost all of the TCR 78 thymocytes (8% of DN
thymocytes) expressed the high level of PD-1 (Fig. 2B). TCR
y8+ cells occupied 16.2 and 49% of the CD44+CD25" and
CD44~CD25- subsets respectively, while CD44+CD25+ and
CD44"CD25+ subsets contained no TCR y8 + cells. The TCR
78 thymocytes (16.2%) in the CD44+CD25~ subset corresponded to PD-1hi9h thymocytes (17%) in this subset (Fig. 1C).
The TCR 78 thymocytes (49%) constitute the majority of the PD-
776
PD-1 expression in early thymocytes
hi
1 9h population (55%) in the CD44-CD25"DN compartment of
normal adult mice (Fig. 1C).
Taken together, the PD-1 low and PD-1h'9h cells in the
CD44+CD25" subset consist of TCR ap NK cells and TCR 78
thymocytes respectively, while most of the PD-1 h ' 9h cells in
the CD44~CD25~ subset are TCR 76 thymocytes. In the TCR
ap non-NK lineage, 10-30% of cells express PD-1 at the low
level from the CD44 + CD25 + and CD44"CD25+ DN subset,
and then the majority of CD44~CD25~ DN cells expressed
PD-1 at the low level.
Augmentation of PD-1 expression by in vivo administration of
the anti-CD3e mAb into normal adult mice
In order to confirm the conclusion that the PD-1 expression
is augmented at the CD44~CD25~ DN stage, in which thymocytes express TCR p chains, we tried to stimulate the CD44"
CD25 + DN thymocytes (60-70 % of the DN compartment) by
anti-CD3 mAb injection. PD-1 expression on DN thymocytes
of normal mice was analyzed 1 day after anti-CD3 mAb (1452C11) administration. The total cell number was reduced to
-1/10 (data not shown), which is mostly due to the reduction
of DP cells (25). Flow cytometric analysis revealed that the
anti-CD3 mAb augmented PD-1 expression levels on DN and
SP cells but not significantly on DP cells. Approximately 54%
of total thymocytes expressed PD-1 at the low or high level
(Fig. 3A). The high level expression of PD-1 was detected on
80-90% of DN cells (Fig. 3A and B) and on 90% of CD8 + SP
cells (data not shown) while only the low level expression of
PD-1 was detected on -40% of CD4 + SP cells and 12.7% of
DP cells (data not shown).
Next we examined in which subsets of DN thymocytes PD1 expression was induced by the anti-CD3 mAb treatment
(Fig. 3C). The total cell number in the DN compartment
did not change significantly (6.0X10 5 versus 6.7X105). The
numbers of CD44~CD25+ DN and CD44"CD25- DN cells were
drastically decreased and increased respectively by the antiCD3 injection (Fig. 1C versus Fig. 3C), which is presumably
due to maturation of CD44-CD25"1" DN cells into CD44~CD25DN cells. Consequently, CD44~CD25~ thymocytes became
the major population, >80% of which expressed PD-1. In
consistency with this result, two-color staining with the antiCD25 and anti-PD-1 mAb revealed that CD25 + PD-r o r l o w DN
cells decreased markedly in contrast to the increase of CD25"
PD-1 + thymocytes (Fig. 3B versus Fig. 1B). Since the total
number of cells in the CD44~CD25~ compartment increased
four times and the fraction of PD-1 + cells doubled, the number
of PD-1 + cells increased almost eight times by 1 day after
anti-CD3 mAb treatment. It is therefore unlikely that the
increase of PD-1 + cells in the CD44-CD25" DN subset by
anti-CD3 stimulation is due to the proliferation of TCR 78 cells
which occupy only 8% of the DN compartment. Taken together,
these results indicate that in vivo anti-CD3 mAb administration
enhanced maturation of DN cells from the CD44"CD25+ DN
stage to the CD44~CD25~ stage, accompanied by augmentation of PD-1 expression.
Augmentation of PD-1 expression on DN cells of RAG-2^'
mice by in vivo administration of the anti-CD3s. mAb
In RAG-2"^ mice, the maturation of thymocytes is blocked at
the CD44"CD25+ DN stage due to the defect of TCR P gene
A) total thymocytes
Total
46
CD4CD8-
13
n
CD8a
PD-1
PD-1
B) DN thymocytes
ft
8Sfv
9.7
CD8a
1
PD-1
CD25
C) DN thymocyte
CD44+CD25+
PD-1
Fig. 3. PD-1 expression on thymocytes from C57BL/6 mice injected
with the anti-CD3 antibody. (A) At day 1 after the anti-CD3 antibody
(145-2C11) injection, total thymocytes from C57BL/6 were analyzed
by three-color flow cytometry with anti-CD4, anti-CD8a and anti-PD1 mAb. The cell number of total thymocytes, especially DP
thymocytes, was reduced to -1/10. (B) DN-enriched thymocytes
were analyzed by two-color staining with anti-CD25 and anti-PD-1
mAb. The purity of DN cells was -99.7%. (C) DN-enriched thymocytes
were analyzed by three-color staining with anti-CD44, anti-CD25 and
anti-PD-1 mAb. The four panels in the bottom right reveal PD-1
expression in the subsets gated in the CD44-CD25 panel. Numbers
in contour profiles indicate the percentage of each population.
rearrangement. The i.p. administration of the anti-CD3 mAb
to RAG-2"'" mice was shown to induce differentiation of DN
thymocytes into DP thymocytes but not into mature CD4 + SP
and CD8 + SP thymocytes (15). PD-1 expression on DN
thymocytes was examined in RAG-2~'~ mice because the
mutant mice have the following advantages: (i) their thymocytes are in less diverged stages of differentiation and (ii)
PD-1 expression can be examined in thymocytes that are
induced to differentiate synchronously by the anti-CD3 antibody administration. In untreated RAG-2"7" mice, 27% of DN
thymocytes expressed the low level but not the high level of
PD-1 in contrast to the wild-type mouse (Fig. 4A). CD44~
CD25 + DN cells were the majority of DN cells in RAG-2~'~
mice (92.2%) because of the differentiation blockage at this
stage. About 37% of CD44 + CD25 + and 32.5% of CD44~
CD25 + cells expressed the low level of PD-1 whereas the
CD44+CD25~ and CD44"CD25" DN subsets contained no
PD-1 expression in early thymocytes
A)
777
A)
O.K
O.I
control
0.1
CD8a
Dayl
B)
4.6
1-6
Day2
Q
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1.6
Day4
CD25
CD8a
B
)
CD4-CD8-
PD-1
CD25
CD4+CD8-
CD4-CD8+
CD2S
CD4+CD8+
Day2
PD-1
Fig 4. PD-1 expression on total thymocytes of RAG-2"'" mice. (A)
Total thymocytes of RAG-2"'" mice were analyzed by flow cytometry
with two combinations (anti-CD4, anti-CD8a and anti-PD-1 mAb or
anti-CD25 and anti-PD-1 mAb). (B) Total thymocytes of RAG-2"'"mice
were analyzed by three-color staining with anti-CD44, anti-CD25 and
anti-PD-1 mAb. About 99% of the total thymocytes were DN cells.
The four panels in the bottom right reveal PD-1 expression in the
subsets gated in the CD44-CD25 panel. Numbers in contour profiles
indicate the percentage of each population.
PD-1 + cells (Fig. 4B). In the profile of CD25 versus PD-1, the
PD-i h| 3 h population was not detected (Fig. 4A versus Fig. 1B).
These data are consistent with the fact that PD-1 + cells in
the CD44 + CD25" and CD44"CD25" subsets belong to the
lineage cells that have accomplished VDJ recombination
such as TCR ccp" NK cells, TCR 78 cells and TCR 0$ thymocytes. Similar results were also obtained from SCID mice
(data not shown).
The high level expression of PD-1 was detected on nearly
98% of total thymocytes 1 or 2 days after the i.p. injection of
anti-CD3 mAb into RAG2~'~ mice (Fig. 5A). The considerable
shift of CD44-CD25+ DN cells into CD44"CD25" DN cells
took place as early as 1 day after the injection of the antiCD3 mAb, probably because of down-regulation of CD25
(Fig. 5A, day 1 CD44 versus CD25). On day 2 after the
treatment, intermediates to DP (CD4 + SP and CD8 + SP)
and DP thymocytes began to emerge. The vast majority of
thymocytes on day 2 were still PD-1 h ' 9h although the expression level of PD-1 was slightly lower than that on day 1. The
total cell number increased 40-50 times on day 4 (1.0X10 6
versus 5.0X107). On day 4 DP cells became the major
population (-80%) of thymocytes with a concomitant decrease
of PD-1 + cells to 15.2% of the total thymocytes. Nearly 80%
of DN and 76.1% of CD8 + SP (intermediates) still expressed
PD-1, while the low level of PD-1 expression was seen in
22% of CD4+SP (intermediates) and 3.6% of DP thymocytes
(Fig. 5B). Two-color staining with the mAb against CD25 and
Day4
•PD-1
Fig. 5. PD-1 expression on thymocytes from RAG-2 ' mice injected
with the anti-CD3 antibody. (A) PD-1 expression on thymocytes was
examined at day 1, 2 and 4 after the administration of 2C11 into
RAG-2"'" mice. Three-color flow cytometry with two combinations
(anti-CD4, anti-CD8a and anti-PD-1 mAb or anti-CD44, anti-CD25
and anti-PD-1 mAb) was performed. As control, mice injected with
an equivalent amount of hamster IgG were examined at day 1.
The cell number increased by -40- to 60-fold and DP thymocytes
accumulated at day 4. (B) PD-1 expression was investigated in
thymocyte subsets in RAG-2"'" mice treated with 2C11 at day 2 and
4. Gates were shown in the CD4-CD8 panels in (A). Numbers in
contour profiles indicate the percentage of each population.
PD-1 revealed that DN cells differentiated sequentially in the
order of CD25+PD-1"/Iow, CD25"PD-1hi9h a n c j CD25-PD-1"
(Fig. 5A). On the other hand, the injection of an equivalent
amount of hamster Ig as a negative control did not change
the distribution of DN subsets or PD-1 expression levels.
These results confirmed the above conclusion that PD-1 is
expressed on the majority of thymocytes at the CD44"CD25"
DN stage preceded by the modest expression on the CD44"
CD25 + DN stage cells.
PD-1 expression in fetal thymocytes.
PD-1 expression in the fetal thymus was analyzed during
normal mouse development since differentiation of DN thymocytes into DP thymocytes occurs in the fetal thymus as
synchronously as in the thymus of anti-CD3 administered
RAG-2"'" mice. Thymocytes positive for the intracellular TCR
P chain appear on day 15 of gestation, while a considerable
number of DP cells appear around days 18 and 19 (16). On
the other hand, TCR yb thymocytes comprise - 3 - 5 % of total
778
PD-1 expression in early thymocytes
CD4~CD8~(DN)
• .2
TCRp rearrangement
Dayl5
1
1 3
CD8a
2
Day 16
ft
f^t
11
CD8a
Differentiation Stage
Fig. 7. A schematic model of PD-1 expression in the transition phase
between DN and DP thymocytes in the TCR a(i lineage. PD-1 can
be expressed at the low level during maturation to CD44~CD25+ DN
thymocytes. Successful TCR (3 rearrangements promote the downregulation of CD25 and the increasing proportion of the PD-1l0W
thymocytes at the CD44~CD25~ DN stage. The expression level
of PD-1 subsequently decreases along with the maturation and
disappears at the DP stage, int, intermediate; DN, CD4~CD8~; DP,
CD4+CD8+.
77
Dayl8
SI
CD8a
4.2
Dayl9
&
*£' 1.9
CD8a
PD-1
Fig. 6. PD-1 expression on fetal thymocytes. Fetal thymocytes at
days 15, 16, 18 and 19 of gestation were analyzed by three-color
staining with anti-CD4, anti-CD8a and anti-PD-1 mAb. The left panels
show the CD4-CD8a profiles of thymocytes. The middle panels show
PD-1 expression on total thymocytes. The right panels show PD-1
expression on the gated subsets in the CD4-CD8a panels. Numbers
in contour profiles indicate the percentage of each population.
thymocytes on days 16-18 of gestation (26,27). In fetal
thymocyte development, the vast majority of PD-1 high thymocytes belonged to the TCR y5 lineage and TCR aB lineage
cells seemed to express the low level of PD-1 on days 1519 of gestation (data not shown). On day 15 of gestation, the
vast majority of thymocytes were fractionated in the DN
compartment, and the low and high levels of PD-1 expression
were detected on 18.1 and 2% of total thymocytes respectively
(Fig. 6). The low level expression of PD-1 was also detected
at the CD44 + CD25 + and CD44+CD25" DN stages (data not
shown). The proportions of the PD-1 low cells in these stages
before the surface expression of TCR B chains were comparable to those in adult RAG-2"'" mice (Fig. 4). On day 16 of
gestation, DP thymocytes appeared and the low and high
levels of PD-1 expression were detected on 13.5 and 3.9%
of total thymocytes respectively. At this stage, 17.5% of DN
and 26.2% of CD8 + SP thymocytes expressed the low level
of PD-1, although only 3.4% of DP cells expressed PD-1. PD1 l o w thymocytes in the CD4~CD8+ fraction seemed to be the
intermediate to the DP stage because they expressed the
low level of TCR cxB (data not shown) (28). On day 18 and
19, DP thymocytes came to constitute 77 and 84% of total
thymocytes respectively, and the percentages of PD-1 low
cells decreased to 9 and 3.9% respectively, because of the
increase of DP thymocytes which barely expressed PD-1. On
day 18 of gestation, 45% of DN and 39% of CD4"CD8+ SP
thymocytes still expressed the low level of PD-1, and on day
19 of gestation, 23% of DN and 27% of CD4"CD8+ SP
thymocytes expressed the low level of PD-1. The CD4~CD8+
SP compartment contained TCR aB low cells but not TCR aBh'9h
cells on days 18 and 19 of gestation (data not shown),
indicating that PD-1 low cells in the CD4"CD8+ fraction were
still the intermediates to the DP stage. On day 19 of gestation,
TCR <xph'9h cells appeared and comprised 60% of the
CD4+CD8~ SP fraction (data not shown) (28), while only 11%
of this fraction expressed the low level of PD-1. This result
indicated that PD-1 would not be expressed on the positively
selected CD4 + SP mature thymocytes. Although further examination should be done for the PD-1 expression on mature SP
cells in T cell development, PD-1 low cells in the CD4 or CD8
SP compartments of day 16-19 of gestation consist mostly of
intermediates to the DP stage. The PD-1 expression pattern
in each subset of thymocytes after birth was similar to that of
adult mice (data not shown). The PD-1 expression is thus
induced in the late DN stage immediately before the transition
phase from DN cells to DP cells not only in the anti-CD3 mAb
stimulated thymus but also in the fetal thymus.
Discussion
PD-1 expression on DN thymocytes was examined in association with expression of the early T cell markers of CD25 and
CD44. About 34% of DN thymocytes expressed PD-1 with
two distinct expression levels (low and high) (Fig. 1B). The
expression level of PD-1 in the differentiation order of the DN
thymocyte subsets was as follows: (i) 13% PD-1|OW and 17%
PD-1 expression in early thymocytes
PD-1hi9h in CD44+CD25"; (ii) 13% PD-1 l0W in CD44 + CD25 + ;
(iii) 28% PD-1 low in CD44-CD25+; (iv) 27.3% PD-1 low and
55% PD-1hi9h in CD44-CD25". PD-1|OW and PD-1hi9h in the
CD44+CD25" DN compartment are likely to be TCR aP NK
cells and TCR y5 cells respectively (Fig. 2). In addition, the
majority of PD-1 h'9h cells in the CD44~CD25- DN compartment
are the TCR yd lineage cells (Fig. 2). In the TCR ap lineage,
the expression of PD-1 started roughly along with the differentiation of thymocytes from the CD44 + CD25 + DN stage and
then the majority of thymocytes at the CD44~CD25~ DN stage
expressed the low level of PD-1.
Recently, the onset of TCR p chain rearrangements was
studied by Godfrey et al. (10). The CD44 + CD25 + subset
represents the germline configuration of the TCR p chain
locus, whereas 75% of CD44~CD25+ thymocytes represent
the rearranged configuration of this locus. CD44~CD25~
thymocytes express mRNA for the TCR p chain. The onset of
TCR p gene rearrangements at the CD44~CD25+ stage is
consistent with the differentiation block at the same stage by
the defect of the DNA rearrangement in RAG"'" and SCID
mice (7,29). The results also imply that TCR p gene rearrangements and signaling through TCR complexes would be
required when thymocytes in the TCR ocp lineage differentiate
beyond the CD44"CD25+ DN stage.
Rough coincidence of the TCR p gene rearrangement
and the increase of PD-1 low population in CD44"CD25" DN
thymocytes led to the hypothesis that augmentation of PD-1
expression would have some relationship to TCR p chain
expression and stimulation through TCR. This hypothesis was
supported by two results. First, neither the low nor the high
level expression of PD-1 was detected on the CD44~CD25~
DN thymocytes in RAG2"/"and SCID mice (Fig. 4A and data
not shown), both of which have defects in VDJ recombination.
In these mice, only -30% of DN thymocytes expressed the
low level of PD-1. Especially, the most mature DN thymocytes
(CD44~CD25~) contained no PD-1 + thymocytes in these mice
(Fig. 4B). Second, the administration of anti-CD3 mAb induced
not only the differentiation of CD44~CD25+ thymocytes into
CD44"CD25" thymocytes but also the high level expression
of PD-1 in RAG2"'~ mice as well as in normal mice (Figs 5A
and 3C). Anti-CD3 antibody stimulation has been shown to
replace signaling through the TCR P chain and pTa heterodimer (15). Only 5% of DP thymocytes expressed the low level
of PD-1, indicating that PD-1 expression is down-regulated
at the DP stage in normal mice. Similar results were obtained
in DP thymocytes of an in vivo anti-CD3 mAb stimulated
RAG2"'" thymus (Fig. 5B, day 4) and of fetal thymus (Fig. 6,
days 16, 18 and 19).
Taken together, PD-1 expression is transiently induced in
the transition phase from the DN stage to the DP stage in the
context of the TCR aP lineage (Fig. 7). The low level expression
of PD-1 is detected at the CD44 + CD25 + and CD44"CD25+
stages, in which TCR p genes have not been rearranged.
After expression of the TCR p chain, the major population
expressed the low level of PD-1 with concomitant CD25 downregulation. Finally, PD-1 expression disappears gradually at
the DP stage. In addition, TCR y8 thymocytes in the DN
compartment, whose maturation process has not been well
defined, express the high level of PD-1 in the adult thymus
as well as in the fetal thymus.
779
We have examined PD-1 expression in adult thymocytes
and peripheral lymphocytes (19). T cells in the thymus and
spleen from adult mice can express PD-1 after in vitro
stimulation with the anti-CD3 antibody, concanavalin A or
phorbol myristate acetate/ionomycin. PD-1 appears to be one
of the activation antigens in peripheral T cells. This view is
consistent with the above conclusion that PD-1 expression
on DN cells occurs at the differentiation stage in which signal
transduction through the TCR complex can take place.
The function of PD-1 in the transition phase between the
DN and DP cells still remains unknown. Further examination
is required for the expression of PD-1 after the DP stage in
association with positive and negative selection. All these
questions will be tested using several genetically manipulated
mice such as PD-1 transgenic mice and PD-1 null mutation
mice which we have recently generated.
Acknowledgements
We thank Dr Ko Okumura for his support for the generation of antiPD-1 mAb (J43). We also thank Dr Shin-ich Nishikawa and Dr
Frederick W. Alt for generous gifts of anti-c-/c/f mAb and RAG-2"'"
mice respectively. We also thank Ms Saeko Okazaki and Ms Reiko
Shinkura for their technical assistance, and Ms Emiko Tadokoro and
Ms Kaori Fukui for their help in preparing the manuscript. This work
was supported by grants from the Ministry of Education, Science,
Sports, and Culture of Japan, and from Yamanouchi Foundation for
Research on Metabolic Disorders.
Abbreviations
DN
DP
PE
RAG
SA
SP
CD4-CD8CD4+CD8+
phycoerythrin
recombination activation gene
streptavidin
single positive
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