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Blood, 15 March 2001, Vol. 97, No. 6, pp. 1765-1775
IMMUNOBIOLOGY
Defective development of NK1.1+ T-cell antigen
receptor   + cells in zeta-associated protein 70 null mice with an accumulation of NK1.1+
CD3 NK-like cells in the thymus
Kazuya Iwabuchi,
Chikako Iwabuchi,
Saori Tone,
Daisuke Itoh,
Noriko Tosa,
Izumi Negishi,
Kazumasa Ogasawara,
Toshimitsu Uede, and
Kazunori Onoé
From the Division of Immunobiology and Molecular
Immunology, Institute for Genetic Medicine, Hokkaido University,
Sapporo, Japan.
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Abstract |
Development of natural killer 1.1+ (NK1.1+)
CD3+ (NK1.1+ T) cells was analyzed in
zeta-associated protein 70 (ZAP-70) null ( /) mice. Both
NK1.1+ TCR  + and NK1.1+
TCR  + cell populations were absent in the thymus and
spleen. By contrast, the number of NK1.1+ CD3
cells was increased in these tissues. The NK1.1+
CD3 thymocytes in ZAP-70/ mice had
surface phenotypes in common with NK or NK1.1+ T cells.
However, some of them were discordant either with NK cells or with
NK1.1+ T cells. The NK1.1+ CD3
cells produced interferon- upon stimulation with NK1.1 cross-linking in the presence of interleukin-2 and exhibited a substantial
cytotoxicity against YAC-1 cells. Moreover, the generation of
NK1.1+ T cells with invariant V14J281 chains was
induced from the NK1.1+ CD3 thymocytes
following stimulation with phorbol myristate acetate and ionomycin in a
neonatal thymic organ culture. An introduction of TCR and transgenes to the ZAP-70/ mice resulted in generation
of an NK1.1+ TCRdim population, whereas
no substantial CD4+ CD8 or CD4
CD8+ population that expressed the introduced
TCR was generated in the mainstream T lineage. These
findings demonstrate that ZAP-70 kinase is indispensable for the
development of NK1.1+ T cells and that the unique
NK1.1+ CD3 thymocytes in
ZAP-70/ mice contain immediate precursors of
NK1.1+ T cells.
(Blood. 2001;97:1765-1775)
© 2001 by The American Society of Hematology.
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Introduction |
The natural killer 1.1+
(NK1.1+) T-cell population is a T-cell subset that
expresses NK markers, ie, NK1.1, and possesses unique biologic
functions.1,2 It has been shown that the
NK1.1+ T cells are selected by ligands on the bone
marrow-derived cells in the thymus,3-5 although we have
demonstrated that the thymic structure also plays an important role in
the positive selection.6 The restriction molecule for most
of the NK1.1+ T-cell population that expresses the
canonical V14J281 has been revealed as CD1d or TL
molecule.7-10 The CD1d molecule presents -galactosylceramide originally derived from marine sponge to NK1.1+ T cells and activates them in the
periphery.11 However, no -galactosylceramide is present
in mammals under normal conditions, and glycosyl-phosphatidylinositol
(GPI) moiety of GPI-anchored antigens appears to be one of the natural
ligands for CD1d-restricted NK1.1+ T
cells.12,13 Although unique functions of the
NK1.1+ T cells have been
demonstrated,1,2,13-19 a number of questions remain
unanswered on the developmental pathway in the thymus and periphery.
Previous studies20,21 demonstrated that T and NK cells were
derived from a common precursor. Although NK1.1+ T cells
may share the developmental pathway with the T and NK cells, it has not
been clear where the NK1.1+ T cells branch off from this
common pathway. In mouse, Ballas et al22 reported that NK
cell function could be detected by gestational day 15 and that
NK1.1+ CD4+ CD8+
double-positive (DP) cells were demonstrable on day 15 to 16 prior to
an appearance of NK1.1 DP thymocytes in the fetal thymic
ontogeny. These authors suggested that the NK1.1+ DP cells
that are transiently detected in the fetal thymus were the progenitors
of NK1.1+ T cells. Meanwhile, the NK1.1 but
not the NK1.1+ T-cell population that expressed both
invariant V14J281 and V8 T-cell receptor (TCR) and exhibited
characteristics of NK1.1+ T cells were present in common
chain-deficient mice.23 Similarly large granular
lymphocytes with a phenotype of CD4 CD8
TCR+ CD16+ NK1.1+
CD45R+ were identified following cultivation of
TCR+ CD4 CD8
NK1.1 thymocytes and splenocytes with interleukin (IL)-2
alone.24 The latter 2 reports support that
NK1.1+ TCR+ cells are generated from the
NK1.1 TCR+ cell population that has already
rearranged the respective TCR gene loci.
Another precursor candidate of NK1.1+ T cells may be
NK1.1+ TCR cell population. Sato et
al25 demonstrated that the NK1.1+ surface
CD3 population could differentiate into mature
NK1.1+ T cells in the presence of IL-15,
granulocyte-macrophage colony-stimulating factor (GM-CSF), and stromal
cells in vitro. This report suggests that the expression of the NK1.1
molecule preceded that of TCR.
Recent studies with genetically engineered mice revealed that the
development of NK1.1+ T cells was defective in
IL-7/, IL-7R/,
IL-2/15R/ ,26 and
CD3 / mice.27 It was also shown with
these engineered mice that development of mainstream T cells was
defective. These findings are consistent with a postulate that the same
or similar molecular mechanisms operate on the development of both
NK1.1+ T cells and mainstream T cells. However,
NK1.1+ T cells as well as TCR+ cells but
not mainstream T cells developed normally in the dominant negative
mutant on the Ras/Raf/Mek/MAPK pathway.28 On the other hand, in T-cell factor-1-deficient mice,29 Fyn-deficient
mice,30 and pre-T/
mice,26,31 the development of NK1.1+ T cells
was selectively abrogated, whereas only minimal defect was observed in
mainstream T cells. These findings suggest that certain signaling
pathways involved in generation of NK1.1+ T cells are
different from those of mainstream T cells.
In the present study, we analyzed development of NK1.1+ T
cells in zeta-associated protein (ZAP)-70/ mice. It was
shown that ZAP-70 tyrosine kinase was essential for the development of
mainstream T cells but not for that of NK cells.32 Indeed,
NK cells differentiated normally in the peripheral lymphoid tissues and
retained the NK activity in ZAP-70/ mice presumably
because p72syk replaced ZAP-70 functions in the NK cell
population. Herein, we demonstrate that development of
NK1.1+ T cells is completely abrogated in
ZAP-70/ mice. Instead, a considerable population of
NK1.1+ CD3 cells was detected in the thymus
as well as in the spleen. Although the surface phenotype of the
NK1.1+ CD3 population was quite unique, this
population retained intact NK functions. Furthermore, it will be shown
that generation of NK1.1+ T cells is induced in the
NK1.1+ CD3 thymocytes following stimulation
with phorbol myristate acetate (PMA) and ionomycin in vitro. A possible
developmental pathway of the NK1.1+ T cells is discussed.
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Materials and methods |
Mice
ZAP-70/ mice32 were provided by
Dennis Y. Loh at the Department of Biology, Nippon Roche Research
Center (Kamakura, Japan). These mice were backcrossed with C57BL/6 (B6)
mice for several generations and maintained in the animal facility at
the Institute for Genetic Medicine, Hokkaido University, in a specific
pathogen-free condition. ZAP-70+/ and
ZAP-70+/+ mice were used as controls for flow cytometric
and functional analyses. ZAP-70//DO10 TCR transgenic
mice were prepared by crossing the ZAP-70/ mice of B6
background with DO10 TCR transgenic mice33 of B10.D2 background. Progenies were screened for the mutant ZAP-70 allele and
the TCR transgene. C57BL/6J-Rag-1tm1rag
(RAG-1/), B6.PL-Thy1a/Cy (B6.
Thy 1.1), and TCR/ and / mice
were purchased from the Jackson Laboratory (Bar Harbor, ME). All mice
were used for experiments at the age of 14 to 20 weeks.
Antibodies and flow cytometry
Thymocytes and spleen cells were first incubated with unlabeled
monoclonal antibody (mAb) 2.4G2 (anti-FcR) to block nonspecific staining and were then stained with a combination of the following mAb
conjugates: biotinylated (biotin)-CD1d (1B1), -CD5 (53-7.3), -CD16
(2.4G2), -CD24 (J11d), -CD25 (7D4), -CD34 (RAM34), -CD44 (IM7), -CD45RB
(23G2), -CD62L (MEL-14), -CD69 (H1.2F3), -Ly49A (A1), -TCR (GL3),
-2B4, and -DX5; fluorescein isothiocyanate (FITC)-CD8 (53-6.7), -CD2
(RM2-5), -CD4 (RM4-5), -TCR (H57-597), -CD117 (2B8), -CD90
(53-2.1), -CD3 (145-2C11), -CD95 (Jo2), -Ly49C (5E6), and -NK1.1
(PK136); and phycoerythrin (PE)-NK1.1, -CD45R (RA3-6B2), and -CD122
(TM-1) (all from Pharmingen, San Diego, CA). A clonotypic mAb for
the transgenic TCR of DO10 mouse, KJ1-26,34 was purified
from hybridoma supernatant with Hi-Trap Protein G (Amersham Pharmacia
Biotech, Uppsala, Sweden) and biotinylated by incubating with
N-hydroxy-succimide biotin (Pierce, Rockford, IL) in
dimethyl sulfoxide solution. Streptavidin-FITC or -Red 670 (Gibco,
Gaithersburg, MD) was used for biotin-mAb. Propidium iodide red
fluorescent dye (Sigma Chemical, St. Louis, MO) was added to the cells
immediately before analysis. Stained cells were analyzed with FACScan
flow cytometer (Becton Dickinson, Mountain View, CA) using CellQuest
software (Becton Dickinson).35
Preparation of NK1.1+ cells
Thymocyes were treated with anti-CD24 and anti-CD8 mAb, and
splenocytes were treated with anti-CD24, CD8, and I-Ab
(1E4)36 mAb at 4°C for 30 minutes. The cells were then
washed twice and resuspended in magnetic beads coated with goat antirat immunoglobulin G (IgG) antibody (Miltenyi Biotec, Bergisch Gladbach, Germany) at 6°C for 15 minutes. Cells that had bound the antibody were depleted by magnetic-activated cell sorting (MACS) system with
VarioMACS (Miltenyi Biotec). Thereafter, the CD24,
CD8, and I-Ab cells were stained
with FITC-anti-TCR mAb and PE-anti-NK1.1 mAb. The stained cells
were sorted into NK1.1+ TCR cells with
FACSVantage (Becton Dickinson). The sorted cells were further cultured
for 4 days in the presence of recombinant human (rh)IL-2 (1000 U/mL;
Pharmaceutical Research Division, Takeda Chemical Industries, Osaka,
Japan) and used for functional analyses.
Cytokine enzyme-linked immunosorbent assay
To evaluate IL-4 production, either unsorted
(1 × 106) or sorted (4 × 104) cells from
the thymus and spleen were stimulated with immobilized anti-CD3 mAb
(145-2C11; Pharmingen) at 10 µg/mL in a total volume of 50 µL RPMI
1640 supplemented with 10% heat-inactivated fetal calf serum, 100 U/mL
penicillin, 100 µg/mL streptomycin, and 5 × 105 M
2-mercaptoethanol in a 96-well flat-bottomed plate for 48 hours. IL-4
in the culture supernatants was quantitated with Cytoscreen Immunoassay
kit for mouse IL-4 (BioSource International, Camarillo, CA) according
to a manufacturer's protocol. To evaluate interferon (IFN)-
production, cells were stimulated with immobilized anti-NK1.1 (PK136;
Pharmingen) at 50 µg/mL in a total volume of 50 µL RPMI-1640 in the
presence of rhIL-2 (1000 U/mL) for 48 hours. IFN- in the culture
supernatants was quantitated with Cytoscreen Immunoassay kit for mouse
IFN- (BioSource International).37
Assay for cytotoxic activity
Cytotoxic activities were evaluated as previously
described.32 In brief, control and ZAP-70/
mice were intraperitoneally administered 200 µg tilorone
(2,7-bis[2-(diethylamino)ethoxy]-9H-fluoren-9-one; Sigma)
per mouse 24 hours before collecting thymocytes and splenocytes. Total
thymocytes or splenocytes depleted of red blood cells were cultured for
7 days in the presence of rhIL-2 (1000 U/mL). The harvested cells were
used as effector cells and incubated with 51Cr-labeled
YAC-1 or P815 cells (5 × 103) at indicated
effector:target ratios for 4 hours.14 Then
51Cr radioactivity released in the supernatant was
quantified with -counter (Auto Gamma 5000; Packard, Canberra,
Australia). Cytotoxicity was expressed as percent specific lysis, which
was calculated as follows: percent specific lysis = [(experimental release  spontaneous release)/(maximum
release spontaneous release)] × 100. Spontaneous release and
maximum release were obtained by incubating target cells alone or with
2N HCl solution, respectively.
Neonatal thymic organ culture
Thymic lobes were obtained from neonatal BALB/c,
RAG-1/, or (B6 × B6.Thy1.1)F1 mice within
24 hours of birth and cultured for 5 to 7 days with medium containing
1.35 mM 2'-deoxyguanosine (dGuo; Nakaraitesque, Osaka, Japan) on
the raft of a membrane filter (0.45 µm; Millipore, Bedford, MA) with
a sterile sponge (Gelfoam; Pharmacia-Upjohn, Tokyo,
Japan).38 The dGuo-treated thymic lobes were transferred
to a Terasaki tissue culture plate (Becton Dickinson, Oxnard, CA) in a
hanging-drop setup with a total volume of 20 µL. Then the sorted
NK1.1 TCR or NK1.1+
TCR thymocytes from ZAP-70/ mice
were seeded (about 5 × 103/lobe to
8 × 103/lobe) and cultured with the thymic lobes of
BALB/c, RAG-1/, or (B6 × B6.Thy1.1)F1 mice
in RPMI-1640-based medium in the presence of 1600 nM PMA (Sigma) and
130 nM ionomycin (Sigma).39,40 Five days later the thymic
lobes were harvested and minced to obtain a single-cell suspension.
Cells were stained with either PE-anti-NK1.1 mAb or PE-control IgG2a
(Pharmingen) and FITC-anti-TCR mAb and analyzed with FACScan as
described above. The proportion of NK1.1+
TCR+ cells was calculated as follows: [percentage of
NK1.1+ TCR+ cells minus percentage of
control IgG2a+ TCR+ cells]. Statistical
analysis was performed according to the Student t test.
Detection of an invariant V chain and RAG-1 transcripts in
induced NK1.1+ TCR+ cells
Total RNA was extracted either from neonatal thymic lobes
obtained from RAG-1/ mice or those cultured with sorted
NK1.1+ TCR cells from
ZAP-70/ thymi in the presence or absence of PMA plus
ionomycin.35 Complementary DNA (cDNA) was synthesized
using random hexanucleotide (Takara Shuzo, Ohtsu, Japan) and Moloney
murine leukemia virus reverse transcriptase (SuperScript; Gibco) at
37°C for 1 hour in the presence of deoxyribonucleoside triphosphates
and ribonuclease inhibitor (RNasin; Promega, Madison, WI). The cDNA
products were used as templates in either ordinary or nested polymerase
chain reactions (PCRs) for amplification of the following gene products
with respective primer pairs (all from Hokkaido System Science,
Sapporo, Japan): V14 Leader/C-rev1 (for first-round PCR),
5'-ATGAAAAAGCGCCTGAGTGCC-3'/5'-CAGGAGGATTCGGAGTCCCA-3'; V14/J281
(for nested PCR),
5-TAAGCACAGCACGTGCACAT-3'/5'-CAATCAGCTGAGTCCCAGCT-3' 9,37,41;
RAG-1 5'/RAG-1 3' (for first-round PCR),
5'-CCAAGCTGCAGACATTCTAGCACTC-3'/5'-CAACATCTGCCTTCACGTCGATCC-3'; RAG-1
5' nest/RAG-1 3' nest (for nested PCR),
5'-CGAAGAAGCACAGAAGGAGAAGG-3'/5'-AAACGATTCCCACAGATGCGGC-3'; and EF-1
5'/3',
5'-CTGCTGAGATGGGAAAGGGCT-3'/5'-TTCAGGATAATCACCTGAGCA-3'.42 Thermal cycling was performed with the following programs: 40 cycles of
heat denaturation at 94°C for 1 minute, annealing at 53°C for
V14 Leader/C-rev1, 52°C for V14/J281, or 50°C for EF-1 5'/EF-1 3' for 1 minute, and elongation at 72°C for 2 minutes. PCR products were electrophoresed on either a 3.0% (for
V14/J281 transcripts) or a 1% (for RAG-1 and EF-1
transcripts) agarose ethidium bromide gel according to the length of
amplified bands.
Analysis of gene rearrangement of TCR V chain from
NK1.1+ TCR thymocytes in
ZAP-70/ mice
Genomic DNA was extracted from either B6 thymocytes, B6 ear
skin, or NK1.1+ TCR thymocytes of
ZAP-70/ mice and subjected to a PCR-based analysis of
gene rearrangements42,43 with slight modifications. PCR was
performed using a primer pair of a coding region of D2 (D2-5')
and 3'-downstream region of J2.7 (J2-3') to detect rearrangements
of D2 to J2 cluster. Rearrangement of V8 to D2J2 was
examined by PCR with a primer pair of a coding region of V8.2
(V8-5') and the same J2 primer as described above. Sequences of
PCR primers were: D2-5', 5'-GTAGGCACCTGTGGGGAAGAAACT-3'; J2-3',
5'-TGAGAGCTGTCTCCTACTATCGATT-3'; and V8-5',
5'-GCATGGGCTGAGGCTGATCCATTA-3'. PCR was performed according to a
standard protocol with 2 units of AmpliTaq Gold DNA polymerase (Roche
Molecular Systems, Branchburg, NJ) with 1 cycle of 8 minutes at 94°C
and 25 cycles of 1 minute at 94°C, 2 minutes at 63°C, and 3 minutes
at 72°C, followed by an extension step of 10 minutes at 72°C. Whole
PCR products were extracted by phenol/chloroform and
ethanol-precipitated and electrophoresed on 1% agarose gel in
Tris-acetate-EDTA (TAE) buffer. Electrophoresed materials were then
transferred to nitrocellulose membrane (Hybond-N+;
Amersham, Little Chalfont, UK) and hybridized with a J2.6 gene segment (5'-CAGCCCTTGCCCTGACTGATT-3') probe labeled with 3'-oligo labeling and detection systems (ECL; Amersham) according to the manufacturer's protocol. Hybridization was performed at 37°C
overnight in a 5 × standard saline citrate 0.02% sodium
dodecyl sulfate-based hybridization solution and washed at room
temperature with 5 × standard saline citrate 0.1% sodium dodecyl
sulfate for 15 minutes once followed by a wash at 37°C for 15 minutes. Then, the membrane was washed several times with
NaCl/Tris-based buffers and incubated with antifluorescein antibody
conjugated with alkaline phosphatase at 4°C overnight with gentle
shaking. The membrane was washed 4 times with 0.4 M NaCl, 0.1 M
Tris-HCl, (pH 7.5), incubated with CDP-Star for 1 minute, and exposed
to x-ray film (Hyperfilm; Amersham).
| |
Results |
Development of NK1.1+ T cells was arrested in
ZAP-70/ mice
To examine development of NK1.1+ T cells in the
ZAP-70/ mice, we performed flow cytometric analysis of
the thymocytes and spleen cells and compared the profiles with those of
control mice (ZAP-70+/ or ZAP-70+/+ mice).
Representative results are shown in Figure
1. NK1.1+ CD3+ or
NK1.1+ TCR+ cells were totally absent in
the thymus of ZAP-70/ mice (0% or 0.02%,
respectively). Instead, a substantial proportion of the
NK1.1+ CD3 or NK1.1+
TCR population was detected (0.42% or 0.39%,
respectively). The NK1.1+ CD3 or
NK1.1+ TCR population was rarely
detectable in the thymus of control mice. When the
TCR+ cell population was compared, no
NK1.1+ TCR+ cells were detected in both
ZAP-70/ mice and control mice. However, a substantial
proportion (0.35%) of TCR+ cells was detected in the
NK1.1 thymocytes of ZAP-70/ mice but a
quite low proportion in those of ZAP-70+/ mice. Similar
results were obtained when spleen cells were analyzed. No
NK1.1+ CD3+ or NK1.1+
TCR+ cell was detected in the spleen of
ZAP-70/ mice, whereas a markedly high proportion of
NK1.1+ CD3 cells was demonstrated in the
ZAP-70/ spleen as compared with that of control mice
(Figure 1).
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| Figure 1.
NK1.1 expression on thymocytes and splenocytes from
control (ZAP-70+/) and ZAP-70/ mice.
Thymocytes and splenocytes were stained with PE-anti-NK1.1 and
FITC-anti-CD3, or FITC-anti-TCR, or with PE-anti-NK1.1 and
biotin-anti-TCR/streptavidin-FITC. Expressions of
NK1.1 (vertical) and CD3, TCR, or TCR (horizontal) are
illustrated. Proportions of NK1.1+ CD3 and
NK1.1+ CD3+ (left column), NK1.1+
TCR and NK1.1+ TCR+
(middle column), and NK1.1+ TCR,
NK1.1+ TCR+, and NK1.1
TCR+ (right column) cells are indicated in the
figures. Results are representative of 6 independent experiments.
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These differences shown in the flow cytometric profiles were again
demonstrated when actual cell numbers were compared between ZAP-70/ mice and control mice (Figure
2). No significant difference in the mean
cell numbers of the thymus and spleen was observed between ZAP-70/ (n = 5) and control mice (n = 6) (thymus:
1.38 ± 0.26 × 108 in ZAP-70/ mice and
1.33 ± 0.32 × 108 in control mice; spleen:
1.75 ± 0.44 × 108 in ZAP-70/ and
1.58 ± 0.37 × 108 in control mice). However, the
number (0.028 ± 0.056 × 105) of NK1.1+
TCR+ cells in ZAP-70/ thymus was
significantly smaller than that of control thymus (7.01 ± 2.56 × 105; P < .001). By
contrast, the number of NK1.1+ TCR
cells in ZAP-70/ thymus was approximately 3- to 4-fold
greater than that of control thymus (ZAP-70/,
2.94 ± 0.46 × 105; control mice,
0.80 ± 0.18 × 105; P < .005).
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| Figure 2.
Cell number of NK1.1+
TCR+, NK1.1+ TCR,
NK1.1+ TCR+, and NK1.1
TCR+ subpopulations in the thymus and spleen of
control and ZAP-70/ mice.
Mean cell numbers of each subpopulation in the thymus and spleen of
control ( , n = 6) and ZAP-70/ ( , n = 5) mice
were calculated and shown as means and SD.
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Although NK1.1+ TCR+ cells were
negligible in both control and ZAP-70/ thymi with the
flow cytometric analysis (Figure 1), the actual number of
NK1.1+ TCR+ cells in control mice was
higher than that in ZAP-70/ mice, as shown in Figure 2
(control, 0.186 ± 0.074 × 105;
ZAP-70/, 0.365 ± 0.163 × 105;
P < .005). On the contrary, the mean cell number of the
NK1.1 TCR+ population was considerably
higher in ZAP-70/ thymus than that in control thymus
(ZAP-70/, 5.79 ± 1.66 × 105; control,
3.29 ± 0.91 × 105; P < .05). These
findings suggest that ZAP-70 is less influential to the development of
TCR+ cells than to that of TCR+ cells.
The marked decrease in the number of NK1.1+
TCR+ cells and significant increase of
NK1.1+ TCR cells were also demonstrated
in the spleen of ZAP-70/ mice (Figure 2). The mean
number of NK1.1+ TCR+ cells in
ZAP-70/ mice was significantly smaller than that of
control mice (ZAP-70/,
0.165 ± 0.176 × 105; control,
10.7 ± 5.75 × 105; P < .05), and the
mean number of NK1.1+ TCR cells in
ZAP-70/ mice was significantly higher than that of
control mice (ZAP-70/,
102 ± 28.6 × 105; control,
47.4 ± 9.90 × 105; P < .05). The small
but substantial population of the NK1.1+
TCR cells seen in control mice might be
attributable to the presence of ordinary NK cells in the spleen. It was
also noted in Figure 2 that the number of NK1.1+
TCR+ cells was considerably smaller in
ZAP-70/ spleen than that in normal spleen
(ZAP-70/,
0.285 ± 0.193 × 105;control,
2.53 ± 1.09 × 105; P < .05), whereas
the number of NK1.1 TCR+ cells in
ZAP-70/ mice was almost the same as that of control
mice (ZAP-70/, 12.6 ± 3.60 × 105;
control, 11.1 ± 2.58 × 105; P = .282).
NK1.1+ CD3 thymocytes in
ZAP-70/ mice exhibited unique phenotype compared with
either ordinary NK cells or NK1.1+ TCR+
T cells
Next, to examine other surface phenotypes of NK1.1+
CD3 cells detected in the thymus of
ZAP-70/ mice using various mAbs that react mainly to T
cells (Figure 3A), mainly NK cells
(Figure 3B), or stem cells (Figure 3C), whole thymocytes obtained from
either control or ZAP-70/ mice were analyzed with flow
cytometry. As shown in Figure 3A, the NK1.1+
population in control mice that corresponds mostly to the
NK1.1+ T cells (Figure 1) was CD1low,
CD4/+, CD5+, CD8,
CD24, CD25, CD44+,
CD90+, and CD122+ as reported in previous
studies.1,2,14,37,44 As far as expressions of CD8, CD24,
CD25, CD90, and CD122 molecules were concerned, NK1.1+
TCR cells in ZAP-70/ mice showed
the same staining pattern as that of NK1.1+ T cells in
control mice. However, these NK1.1+ TCR
cells expressed neither CD4 nor CD5 molecules but showed higher CD44
(Figure 3A), CD2, and CD16 (Figure 3B) fluorescence intensity than
NK1.1+ T cells in control mice. When markers for the
precursor population were analyzed, both NK1.1+
TCR thymocytes of ZAP-70/ mice and
the thymic NK1.1+ T cells of control mice were
CD117 and CD34 (Figure 3C).
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| Figure 3.
Phenotype of NK1.1+ thymocytes from control and
ZAP-70/ mice.
Thymocytes were stained with PE-anti-NK1.1 or FITC-anti-NK1.1 and
various cell surface markers as described in "Materials and
methods." Expressions of NK1.1 (vertical) and other markers
(horizontal) on thymocytes are illustrated: expressions of NK1.1 and
T-cell surface markers (A), NK surface markers (B), and stem cell
markers (C). Results are representative of 6 independent experiments.
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In addition, the NK1.1+ thymocytes in
ZAP-70/ mice were CD45R+,
CD45RB+, CD62L+, CD69int,
CD95, Ly49A, Ly49C, and
2B4+, whereas the NK1.1+ T cells of control
mice were CD2+, CD45R, CD45RB,
CD62L, CD69/low,
CD95+, Ly49A/+, Ly49C/+, and
2B4 (Figure 3B). These profiles of NK1.1+
thymocytes in ZAP-70/ were similar to those of ordinary
NK cells in the peripheral lymphoid organs.
We then examined surface expression of a pan-NK marker,
DX5,45 on NK1.1+ TCR cells in
the thymus and spleen of ZAP-70/ mice and compared it
with that of NK1.1+ cells in control mice. As shown in
Figure 4, approximately 96% of
NK1.1+ TCR cells in ZAP-70/
thymus expressed DX5, whereas most NK1.1+ TCR+
cells expressed no DX5 in ZAP-70+/ thymus. In the
ZAP-70+/ spleen, however, approximately 30% of
NK1.1+ TCR+ cells expressed DX5. This
finding suggests a phenotypic difference between NK1.1+ T
cells in the thymus and those in spleen of normal mice. Almost the same
proportions expressed DX5 in NK1.1+ TCR
cells of both in ZAP-70+/ and ZAP-70/
spleens.
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| Figure 4.
DX5 expressions on NK1.1+ thymocytes and
splenocytes from control (ZAP-70+/) and
ZAP-70/ mice.
Thymocytes and splenocytes were stained with PE-anti-NK1.1,
FITC-anti-TCR, and biotinylated anti-DX5 followed by
streptavidin-Red 670. NK1.1+ TCR+ or
NK1.1+ TCR cells in the thymus and
spleen were electronically gated and analyzed for the expression of DX5
on FACScan. Dead cells were electronically gated out with propidium
iodide staining. FACS profiles were shown in contour with NK1.1 versus
DX5 staining.
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Then, to compare the surface phenotype of NK1.1+
TCR thymocytes in ZAP-70/ mice with
NK1.1+ TCR cells of B6 and another
NKT-deficient strain, RAG-1/, CD8
HSA thymocytes of ZAP-70/ mice,
CD8 HSA CD3 thymocytes from
B6 mice, and whole thymocytes of RAG-1/ mice were
analyzed for the various surface markers (Figure
5). These populations were enriched for
the NK1.1+ CD3 cells and enabled us to
compare the surface phenotypes more precisely. Figure 5A shows that
CD44, CD45RB, CD69, and CD16 are more highly expressed on the
NK1.1+ CD3 cells of ZAP-70/
or RAG-1/ mice than those from B6 mice. The higher
expressions of these molecules on the cells of 2 NKT-defective mutants
could not be explained with the cell sizes, because the
NK1.1+ CD3 thymocytes from 3 kinds of mice
showed almost the same values of forward light scatter. Figure 5B,C
shows that 2B4, CD62L, or CD45R is expressed on NK1.1+
CD3 thymocytes from ZAP-70/ and
RAG-1/ mice but not on those of B6 mice. On the other
hand, approximately 25% to 35% of ordinary NK cells of B6 mice were
either Ly49A+ or Ly49C+.46 No
expressions of Ly49A and Ly49C were detected on NK1.1+
CD3 cells of ZAP-70/ and
RAG-1/ mice. It was also noted that the expression of
CD95 was lower on NK1.1+ CD3 thymocytes of
ZAP-70/ and RAG-1/ than that of B6
mice.
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| Figure 5.
Phenotype of NK1.1+CD3
thymocytes from ZAP-70/, RAG-1/, and
B6.
Whole thymocytes (B6-RAG-1/), thymocytes enriched for
CD8 HSA population with MACS
(ZAP-70/), and thymocytes enriched for
CD8 HSA with MACS and electronic gating for
CD3 thymocytes (B6 mice) were stained with PE-anti-NK1.1
and various cell surface markers (grouped in panels A-C) as described
in "Materials and methods." Expressions of NK1.1 (vertical) and
other markers (horizontal) on either enriched or whole thymocytes are
indicated.
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These results are summarized in Table 1.
This table demonstrates that the NK1.1+ CD3
cells in the thymus of ZAP-70/ mice share largely
common features with ordinary NK cells. However, the
NK1.1+CD3 cells exhibit several distinct
phenotypes that represent neither NK1.1+ T cells nor NK
cells. In this table, it is of note that
NK1.1+CD3 cells with surface phenotype
similar to those of ZAP-70/ mice are present in
RAG-1/ thymus.
NK1.1+ CD3 thymocytes in
ZAP-70/ mice produced IFN- upon stimulation with
NK1.1 cross-linking in the presence of IL-2
To examine functions of the NK1.1+
TCR cells in the thymus of ZAP-70/
mice, we analyzed the ability to produce cytokines. It has been shown
that NK1.1+ T cells produce large amounts of IL-4 and
IFN- shortly after stimulation with CD3
cross-linking.13,48,49 When thymocytes or splenocytes
obtained from either control or ZAP-70/ mice were
stimulated with immobilized anti-CD3 mAb for 48 hours, these cells
from control ZAP-70+/ mice produced considerable amounts
of IL-4 and IFN-. By contrast, neither IL-4 nor IFN- was produced
from thymocytes or splenocytes of ZAP-70/ mice (data
not shown). This finding indicates again that NK1.1+ T
cells are absent in the thymus and spleen of ZAP-70/
mice and that TCR+ cells present in these populations
are unable to quickly respond to the CD3 cross-linking.
We then stimulated thymocytes from control or ZAP-70/
mice with immobilized anti-NK1.1 mAb in the presence or absence of
rhIL-2. Thymocytes from ZAP-70/ mice produced
substantial amounts of IFN- upon stimulation with NK1.1
cross-linking in the presence of rhIL-2 (data not shown). To quantify
IFN--producing ability of NK1.1+
TCR cells, we sorted the NK1.1+
TCR cells from thymi and spleens of
ZAP-70/ mice as well as from spleens of control mice.
These cells were then stimulated with immobilized anti-NK1.1 mAb in the
presence of rhIL-2 (Figure 6). The
NK1.1+ TCR thymocytes and spleen cells
of ZAP-70/ mice produced considerable but
slightly lower amounts of IFN- than that produced by the splenic NK
cells of control mice.
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| Figure 6.
Production of IFN- by thymocytes and
splenocytes from control and ZAP-70/ mice.
Sorted NK1.1+ TCR splenocytes
from control mice (open bar), NK1.1+
TCR splenocytes (shaded bar), and
NK1.1+ TCR thymocytes (closed bar) from
ZAP-70/ mice were cultured in medium alone (medium) or
with immobilized anti-NK1.1 mAb (NKR-P1) in the presence of rhIL-2
(1000 U/mL). Then, IFN- in the culture supernatants were quantified
by enzyme-linked immunosorbent assay. Results are representative
results of 3 separate experiments.
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NK1.1+ CD3 thymocytes in
ZAP-70/ mice showed an intact cytotoxic
activity
To examine cell-mediated functions of the NK1.1+
TCR cells in ZAP-70/ mice,
cytotoxic activity was then analyzed. Thymocytes and splenocytes from
either control or ZAP-70/ mice that had been
administered tilorone were cultured with rhIL-2 for 7 days.14,32 The harvested cells were then analyzed for the
killing activity against 51Cr-labeled YAC-1 or P815 cells.
As shown in Figure 7, both thymocytes and
splenocytes from either control or ZAP-70/ mice showed
significant cytotoxicity against YAC-1 cells but not against P815
cells. The cytotoxicities seen in both thymocytes and spleen cells of
ZAP-70/ mice were consistently higher than those seen
in control mice. This result was consistent with the increased
proportions of NK1.1+ CD3 cells in the thymus
and spleen of ZAP-70/ mice as compared with those of
control mice (Figure 1).
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| Figure 7.
Cytotoxicity of thymocytes and splenocytes from control
and ZAP-70/ mice.
Thymocytes and splenocytes from control ( , ) and
ZAP-70/ ( , ) mice administered tilorone 24 hours
before collecting cells were cultured for 7 days in the presence of
rhIL-2 (1000 U/mL). Cells were harvested and cultured with
51Cr-labeled YAC-1 (, ) or P815 (, ) cells at
indicated effector:target ratios for 4 hours. Percent specific lysis
was calculated as described in "Materials and methods."
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|
When the harvested cells from thymocyte cultures at day 7 were analyzed
for NK1.1 and TCR expressions with flow cytometry, almost 100%
of cells were CD3 among NK1.1+ cells of
ZAP-70/ mice, whereas 40% and 60% of cells were
CD3 and CD3+, respectively, among
NK1.1+ cells of ZAP-70+/ mice, as reported
previously.14 Similarly, when harvested spleen cells were
analyzed, 100% of NK1.1+ cells were CD3 in
ZAP-70/ mice, whereas 70% and 30% of the
NK1.1+ cells were CD3 and CD3+,
respectively, in ZAP-70+/ mice (data not shown). These
findings suggest that no proportional change of CD3 and
CD3+ populations was induced during the culture with IL-2.
NK1.1+ TCR thymocytes had a
germline configuration in TCR gene locus
Flow cytometric analyses demonstrated that NK1.1+
TCR cells expressed neither TCR nor CD3 molecules
on the cell surface. To examine a configuration of TCR gene, the
rearrangement of TCR gene in NK1.1+ CD3
cells obtained from the thymus of ZAP-70/ mice and
thymocytes and skin cells of C57BL/6 mice (control) was analyzed with a
PCR-based technique. The rearrangement of V8 to D2J2 was not
detected in the TCR gene locus of the NK1.1+
TCR cells from ZAP-70/ mice (data
not shown). In addition, we found no band being generated by D2 to
J2 rearrangements in the NK1.1+ TCR
cells of ZAP-70/ mice (Figure
8). Thus, it was demonstrated that the
TCR gene locus of the NK1.1+ TCR
cells from ZAP-70/ mice was in a germline
configuration.
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| Figure 8.
Gene rearrangement on TCR loci in NK1.1+
TCR thymocytes.
A PCR was performed with genomic DNA from B6 thymocytes (Thy; left
lane), B6 ear skin (middle lane), or the
NK1.1+CD3 thymocytes of
ZAP-70/ mice (right lane) with a primer pair of a
coding region of D2 (D2-5') and 3'-downstream region of J2.7
(J2-3') to detect rearrangements of D2 to J2 cluster as
described in "Materials and methods." Germline bands (G) and a
rearranged band (R) are indicated.
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|
Generation of NK1.1+ TCR+ cells was
induced from NK1.1+ TCR thymocytes of
ZAP-70/ mice in hanging-drop culture with PMA
and ionomycin
To directly examine the developmental potential of
NK1.1+ TCR thymocytes in
ZAP-70/ mice, a devised induction assay of
NK1.1+ TCR+ cells was performed. The
NK1.1+ TCR cells were sorted from the
thymocytes of ZAP-70/ mice (Figure
9A, left panel) and cultured with PMA and
ionomycin in dGuo-treated neonatal thymi from BALB/c mice in a
hanging-drop setup. As shown in Figure 9A (middle and right panels), a
substantial proportion (3.03%) of NK1.1+
TCR+ cells was detected after 5 days of culture.
BALB/c mice express no NK1.1 antigen. Thus, rearrangement of TCR genes
appeared to be induced in the NK1.1+
TCR population of ZAP-70/ mice by
an addition of PMA plus ionomycin. When NK1.1+
TCR cells of ZAP-70/ mice were
cultured with thymi of RAG/ mice in the presence of PMA
plus ionomycin, small but substantial numbers of NK1.1+
TCR+ cells were induced (data not shown). No
NK1.1 TCR+ cells were detected
in this setup. In addition, NK1.1+ TCR+
cells induced in the thymic organ culture of (B6 x
B6.Thy1.1)F1 mice were Thy1.1 (data not
shown). These findings again indicated that the induced NK1.1+ TCR+ cells were derived from
NK1.1+ TCR cells of ZAP-70
/ mice, and large NK1.1
TCR+ populations seen in Figure 9A (middle and right
panels) appeared to be derived from the dGuo-treated thymi of
BALB/c mice.
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| Figure 9.
Generation of NK1.1+ TCR+
cells from NK1.1+ CD3 thymocytes of
ZAP-70/ mice in neonatal thymic organ culture.
(A) A representative flow cytometric profile of inductive generation of
the NK1.1+ TCR+ cells in the dGuo-treated
thymi in the presence of PMA plus ionomycin. The sorted
NK1.1+ TCR cells for the culture were
demonstrated in the square of the left panel. Proportions of sorted
NK1.1+ TCR cells were 98.8% to 99.5%.
Collected cells from cultures were stained with either PE-mouse IgG2a
(isotype control of PK136)/FITC-anti-TCR/propidium iodide
(middle panel) or PE-anti-NK1.1/FITC-anti-TCR/propidium iodide
(right panel) and analyzed with FACScan. The proportions of
NK1.1+ TCR+ cells are indicated. (B) Mean
proportion of NK1.1+ TCR+ cells. The
NK1.1+ CD3 (about 5 × 103/lobe
to 8 × 103/lobe) were seeded to the dGuo-treated
neonatal thymic lobes and cultured in hanging-drop setup in the absence
(left column) or presence (right column) of PMA and ionomycin. Five
days later, total cells were stained as described for panel A. The net
proportion of induced NK1.1+ TCR+ cells
were calculated as follows: [percentage of NK1.1+
TCR+ cells minus percentage of control
IgG2a+ TCR+ cells]. The data indicate
mean net proportion and SD.
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|
Figure 9B summarizes 4 separate experiments with BALB/c thymi.
Significant generation of NK1.1+ TCR+
cells was induced in the presence of PMA plus ionomycin as compared with those cultured in the absence of PMA plus ionomycin
(P < .05). A small population of NK1.1+
TCR+ cells seen in the cultures without PMA plus
ionomycin could not be explained.
When NK1.1 CD3 thymocytes from
ZAP-70/ mice were cultured in the hanging-drop setup,
substantial proportions of both NK1.1+
TCR+ and NK1.1+ TCR
cells were detected (data not shown). This finding suggests that the
cells in the NK1.1 TCR population
differentiate to NK1.1+ TCR+ cells
through NK1.1+ TCR stage.
Invariant V14J281 and RAG-1 transcripts were detected in the
induced NK1.1+ TCR+ cells
We then analyzed TCRV usage in NK1.1+
TCR+ cells that had been generated from
NK1.1+ TCR cells of
ZAP-70/ mice following culture with the neonatal thymi
in the presence of PMA plus ionomycin. In this experiment the neonatal
thymi were obtained from RAG-1/ mice of B6 background
to exclude a possibility that V14J281 transcripts were derived
from the thymi. Reverse transcriptase (RT)-PCR was then performed using
total RNA extracted from the cultured NK1.1+
TCR cells. As shown in Figure
10 (lane 3), the V14J281
transcripts were detected in the thymic lobes in which sorted
NK1.1+ TCR cells were cultured with PMA
plus ionomycin. The V14J281 transcripts were detected neither in
the culture of thymic rudiments alone nor in the NK1.1+
TCR thymocytes cultured without PMA plus ionomycin
(Figure 10, lanes 1,2). Thus, the NK1.1+
TCR+ cells induced from the NK1.1+
TCR population by PMA plus ionomycin expressed
V14J281 transcripts. When concomitant expressions of RAG-1 were
examined in the organ cultures, RAG-1 expression was clearly detected
in the culture where the band of invariant V was amplified (Figure
10, lane 3). A very faint band was detected in the culture without PMA
plus ionomycin (lane 2).
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| Figure 10.
Detection of V14J281 transcripts with RT-PCR in
inductive cultures.
Total RNA was extracted from the thymic lobes (RAG-1/
in C57BL/6 background) alone (lane 1) or lobes with sorted
NK1.1+ TCR cells in the presence (lane
3) or absence (lane 2) of PMA plus ionomycin, and RT-PCR was performed
as described in "Materials and methods" with primer pairs V14
Leader/C-rev1 and V14/J281, RAG-1 5'/3' and RAG-1 5' nest/3'
nest, or EF-1 5'/EF-1 3' for positive control. RT-PCR was also
performed without RNA (lane 4) as control. Results are representative
of 3 separate experiments.
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NK1.1+ TCRdim+ thymocytes were generated in
ZAP-70//DO10 TCR transgenic mice
The present results suggested that ZAP-70 deficiency was directly
associated with lack of rearrangement of TCR genes in the NK1.1+ TCR population. We then asked
whether an introduction of rearranged TCR and genes induced
generation of a particular NK1.1+ TCR+
population in the ZAP-70/ background. To examine this
possibility, we crossed ZAP-70/ mice with DO10 TCR
transgenic mice and analyzed the generation of NK1.1+
TCR+ cells in the thymus of
ZAP-70//DO10 mice. As seen in Figure
11A, the NK1.1+ thymocytes
of ZAP-70//DO10 mouse expressed a substantial level of
TCR molecules compared with those in ZAP-70/
thymus. The level of TCR expression, however, was low compared with that of an NK1.1+ TCR+ population in
ZAP-70+/ mice. When these thymocytes were stained with a
clonotypic antibody, KJ1-26, it was shown that an NK1.1+
KJ1-26dim population was present in the thymus (Figure
11B). The total RNA was then extracted from either the sorted
NK1.1+ KJ1-26dim or NK1.1
KJ1-26+ cells, reverse-transcribed, and PCR-amplified with
specific primers for VDO and JDO. Figure
11B shows that VDOJDO messages are present in both NKT and T-cell populations. Thus, it seemed that the
lack of ZAP-70 showed no significant influence on the expression of
rearranged TCR gene in the NK1.1+ CD3
population. The NK1.1+ TCRdim population
expressed no CD4 but broader ranges of CD8 molecules (Figure 11A).
Notably, Figure 11Aiv shows that development of the ordinary thymocyte
was still arrested at the DP stage irrespective of the introduction of
TCR and transgenes in ZAP-70//DO10
mouse.
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| Figure 11.
Detection of NK1.1+ TCRdim thymocytes in
ZAP-70//DO10 TCR transgenic mouse.
(A) Thymocytes obtained from ZAP-70+/,
ZAP-70/, or ZAP- 70//DO10 mice were
stained with PE-anti-NK1.1/FITC-anti-TCR (i),
PE-anti-NK1.1/FITC-anti-CD4 (ii), PE-anti-NK1.1/FITC-anti-CD8
(iii), and PE-anti-CD4/FITC-anti-CD8 (iv), and analyzed. Proportions
of NK1.1+ TCR+, NK1.1+
TCR (upper and middle panels), or
NK1.1+ TCRdim populations (lower panel)
are indicated in the leftmost panels on the top of each squared region.
Results are representative of 3 separate experiments. (B) Flow
cytometric analysis and RT-PCR detection of transgenic TCR in
NK1.1+ TCRdim thymocytes in
DO10/ZAP-70/ mice. Thymocytes of
DO10/ZAP-70/ mice were stained with PE-anti-NK1.1
antibody and biotinylated KJ1-26 followed by streptavidin-FITC. Dead
cells were electronically gated out with propidium iodide staining. The
NK1.1+ TCRdim thymocytes (NKT) and
NK1.1 TCR+ thymocytes (T) (demarcated
with squares in the left panel) were sorted, and the expressions of
DO10-specific TCR chain (VJDO) and C were
examined with RT-PCR.
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| |
Discussion |
It has been shown that ZAP-70 tyrosine kinase is essential for
development of mainstream T cells but not for NK cells.32 Targeted disruption of ZAP-70 led to complete blockade of the development of mature-type T cells both in the thymus and in the periphery. By contrast, the development of ordinary NK cells was intact
in the ZAP-70/ mice, although ZAP-70 was also expressed
in the NK cells.50 Thus far, the lineage relationship
among T, NK, and NK1.1+ T cells has been unclear.
In the present study, we examined development of the NK1.1+
T cells in the ZAP-70/ mice and found an arrested
development of NK1.1+ CD3+ cells in both thymus
and spleen. Neither NK1.1+ TCR+ nor
NK1.1+ TCR+ cells were detected in the
thymus and spleen. The absence of the NK1.1+
TCR+ population was not due to the defective
development of the TCR lineage T cells per se, because
significantly large populations of NK1.1
TCR+ cells were detected in the thymus and spleen of
ZAP-70/ mice as compared with those in control mice. It
was reported that the NK1.1+ TCR+ cell
population was expanded in CD3/ mice.27
Thus, the gene disruption of chain and ZAP-70 led to different
developmental defects in NK1.1+ TCR+
cells, even though ZAP-70 is located downstream of chain. This difference should be pursued in further studies.
Interestingly, we found markedly increased numbers of
NK1.1+ TCR cells in the thymus of
ZAP-70/ mice. The surface phenotype of the
NK1.1+ TCR cells summarized in Table 1
suggests that the NK1.1+ TCR cells
belong to a unique sobpopulation different from ordinary NKT or NK
cells. Approximately 25% to 35% of splenic NK cells express either
Ly49A or Ly49C in the H-2b background.46
However, the NK1.1+ TCR thymocytes of
ZAP-70/ mice (H-2b background) express
neither Ly49A nor Ly49C. The expression of Ly49 on NK cells is
developmentally regulated in a nonrandom manner.46 Thus,
the present findings suggest that the NK1.1+
TCR thymocytes are of distinct cell type or may
belong to a precursor population of the NK1.1+ T cells.
Indeed, we could show that the latter might be the case. The
NK1.1+ TCR thymocytes stimulated by an
addition of PMA plus ionomycin in the thymic organ culture developed
into NK1.1+ TCR+ cells.
Interestingly, the NK1.1+ CD3 thymocytes of
B6-RAG-1/ showed almost superimposable phenotypes to
those of ZAP-70/ mice. Thus, developmental defects that
lead to TCR gene rearrangement may generate accumulation of cells of
the same type. However, the TCR gene disruption on or locus
exerted somehow differential influences on the development of
NK1.1+ T cells. In the TCR/ mice, an
accumulation of NK1.1+CD3 thymocytes was
observed (data not shown). On the other hand, NK1.1+
TCR+ but not NK1.1+ CD3
thymocytes were detected in TCR/ mice51
(data not shown). Thus, it seems important to elucidate genes and
signals vital on certain stages of the development of NK1.1+ T cells in further studies.
In normal B6 mice, a small population of
NK1.1+CD3 thymocytes were detectable, and it
was demonstrated that these NK1.1+D3 cells
were phenotypically ordinary NK cells (Figure 5). This finding,
however, does not exclude a possibility that some of these
NK1.1+ thymocytes may correspond to a putative precursor
population seen in ZAP-70/ mice.
When the NK1.1+ TCR cells were analyzed
in the spleen of ZAP-70/ mice, the number of the
NK1.1+ TCR cells also increased as
compared with that of control mice. We reasoned that the putative
precursor population for the NK1.1+ T cells that could not
be readily distinguished from ordinary NK cells might also be present
and both the precursor and ordinary NK cell populations were recognized
with the increasing number of NK1.1+
TCR cells in the spleen of ZAP-70/
mice. Although expressions of CD45R, CD62L, and 2B4 molecules were
relative characteristics of NK1.1+ TCR
thymocytes in ZAP-70/ mice (Figure 5), these molecules
could be induced on the splenic NK cells upon activation. Thus far, no
appropriate markers that can definitely distinguish NK cells and the
precursor cells in the spleen are available. The precise populations
that make up the NK1.1+ TCR cells in
the spleen of ZAP-70/ mice should be examined in
further studies to clarify whether the NK1.1+
TCR population in the spleen indeed contains the
precursors of NK1.1+ T cells.
We demonstrated that substantial natural cytotoxicity and IFN-
production upon stimulation via NKR-P1 molecules were demonstrated in
the thymic NK1.1+ TCR cells of
ZAP-70/ mice. It was shown that a putative precursor
population of NK cells in bone marrow expressed CD45R and NK1.1
molecules and exhibited cytotoxicity against YAC-1 target
cells.52 Eberl and MacDonald53 reported that
the same population in bone marrow with surface markers similar to
NK1.1+ TCR thymocytes in
ZAP-70/ mice contained precursors for
NK1.1+ T cells. Thus, it seems to us that the putative
precursor population (NK1.1+ TCR cells)
for either NK or NK1.1+ T cells first acquires NK functions.
The NK1.1+ TCR thymocytes in
ZAP-70/ mice showed a germline configuration in TCR
gene. After 5 days of culture with PMA plus ionomycin, the
NK1.1+ TCR+ cells were detected and the
canonical V14J281 transcripts were clearly demonstrated. Sato et
al25 demonstrated that a pre-NKT cell population expressing
NK1.1, TCR, pre-T, and RAG1/2 could differentiate into
mature CD3+ V14+ NKT cells in the presence
of IL-15, GM-CSF, and bone marrow-derived stromas. Because the TCR
genes were not rearranged in the NK1.1+
TCR thymocytes of ZAP-70/ mice,
these cells may be antecedents for the pre-NKT. However, these results
appeared to be in discordance with the stepwise model proposed by
DiSanto and Rodewald26 for the development of
NK1.1+ T cells. Using chain/ mice,
these authors demonstrated that the NK1.1+ T cells
differentiated from T cells that expressed V14 and V8 chains.
These precursor cells possessed characteristic profiles in cytokine
productions but expressed no NK1.1 or Ly49 antigens. The difference may
be attributable to the mice studied but should be elucidated in further investigation.
NK1.1+ TCR cells seen in
ZAP-70/ mice lacked both CD34 and CD117 expressions. It
was reported that fetal thymic NK1.1+ CD117+
and NK1.1 CD117+ cells at gestational day 15 were capable of generating mainstream T cells, NK1.1+ T
cells, and NK cells, whereas NK1.1+ CD117
cells remained CD4 CD8 and committed
exclusively to NK cells.54 Although the NK1.1+
TCR cells in the thymus of ZAP-70/
mice or pre-NKT cells reported by Sato et al25 resemble the NK1.1+ CD117 cells in the fetal thymus, the
NK1.1+ TCR cells and pre-NKT cells
could differentiate into the NK1.1+ T cells in the presence
of PMA plus ionomycin or IL-15 plus GM-CSF in conjunction with stromal
cells from either thymus or bone marrow, respectively.
The TCR gene loci of NK1.1+ TCR
cells in ZAP-70/ mice were in germline configuration.
Because the disruption of ZAP-70 kinase led to no defect in TCR gene
rearrangement in mainstream T cells,32,33 the mechanism
underlying the lack of TCR expression on the NK1.1+
TCR cells was unclear. In the last experiment, we
showed that introduction of rearranged TCR genes to
ZAP-70/ mice resulted in generation of an
NK1.1+ TCRdim population. Thus,
disruption of ZAP-70/ might influence on the TCR
rearrangement but not on the expression of the rearranged TCR genes.
Perhaps the TCR rearrangement in NK1.1+ T-lineage cells is
more ZAP-70-dependent than that in mainstream T cells, which cannot be
compensated by Syk kinases.
We show herein a unique NK1.1+ CD3 cell
population in the thymus of ZAP-70/ mice, and this
population may contain a precursor for NK1.1+ T cells and
possesses intact NK cell functions. It seems that NK1.1+ T
cells and NK cells share a critical pathway for their differentiation. Detailed single-cell-based analyses of the generation of
NK1.1+ T cells from NK1.1+
TCR thymocytes but not from minor contaminants in
the dGuo-treated thymic lobes with PMA plus ionomycin are undertaken in
our laboratory.
| |
Acknowledgments |
We wish to thank Dr D. Y. Loh for providing us with the
ZAP-70/ mouse, Drs T. Matsushita and M. Hosokawa
for reagents, and the Pharmaceutical Research Division of Takeda
Chemical Industries for IL-2. We also thank Ms Ryoko Hosohata and Ms
Kaori Kohno for their secretarial assistance with the manuscript.
| |
Footnotes |
Submitted March 31, 2000; accepted November 6, 2000.
Supported by a Grant-in-Aid for Scientific Research (B, C) from the
Ministry of Education, Science, Sports and Culture, Japan; a Research
Grant for Immunology, Allergy and Organ Transplant and a Research Grant
for Aging from the Ministry of Health and Welfare, Japan; Grants from
the Hokkaido Foundation for the Promotion of Scientific and Industrial
Technology; the Tomakomai East Hospital Foundation; the Nishimura Aging
Fund; and the Itoh Foundation for the Promotion of Medical Sciences.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Kazunori Onoé, Division of Immunobiolgy,
Institute for Genetic Medicine, Hokkaido University, Kita-15 Nishi-7,
Kita-Ku, Sapporo 060-0815, Japan; e-mail: kazunori{at}imm.hokudai.ac.jp.
| |
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1.
MacDonald H.
NK1.1+ T cell receptor-/+ cells: new clues to their origin, specificity, and function.
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Abstract
Introduction