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*
Division of Immunology and Rheumatology, Department of Medicine, Stanford Medical School, Stanford, CA 94305;
Department of Hematology and Oncology, Beth Israel Deaconess Medical Center, and
Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02215; and
§
CREST and Department of Molecular Immunology, Chiba University Graduate School of Medicine, Chiba, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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281-/- mice as compared with wild-type
mice. CD8+NK1.1+ T cells were not clearly
detected, even in the wild-type mice. In bone marrow from the wild-type
mice, CD8+NK1.1+ T cells were easily detected,
about twice as numerous as CD4+NK1.1+ T cells,
and were similar in number to
CD4-CD8-NK1.1+ T cells. All three
marrow NK1.1+ T cell subsets were reduced about 4-fold in
CD1-/- mice. No reduction was observed in
CD8+NK1.1+ T cells in the bone marrow of
J281-/- mice, but marrow
CD8+NK1.1+ T cells were markedly depleted in
ß2m-/- mice. All NK1.1+ T cell
subsets in the marrow of wild-type mice produced high levels of
IFN-
, IL-4, and IL-10. Although the numbers of marrow
CD4-CD8-NK1.1+ T cells in
ß2m-/- and
J281-/- mice were similar to those in
wild-type mice, these cells had a Th1-like pattern (high IFN-, and
low IL-4 and IL-10). In conclusion, the large majority of
NK1.1+ T cells in the bone marrow are CD1 dependent. Marrow
NK1.1+ T cells include CD8+,
V14-J281-, and
ß2m-independent subsets that are not clearly detected in
the thymus. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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II), and asialo-GM1, with NK cells (1, 2). Their
identity as T cells is indicated by expression of CD3-TCR complex, the
very early T cell activation Ag CD69, and low levels of L-selectin
(CD62L) (1). Unlike conventional T cells,
NK1.1+ T cells have displayed a highly restricted
TCR repertoire, which consists of an invariant
V14-J281 -chain
paired preferentially with polyclonal Vß8.2,
Vß7, or Vß2 chains
(3, 4). This highly skewed TCRß repertoire is
positively selected by the nonpolymorphic MHC class I-like CD1d
molecule in association with ß2-microglobulin
(ß2m)3 (1, 5, 6, 7, 8, 9). It has been reported that NK1.1+ T cells
recognize CD1d-bound glycolipid ligands, such as glycosyl
phosphatidylinositol and ceramides (10, 11).
NK1.1+ T cells rapidly produce large amounts of IL-4 upon
in vivo administration of anti-CD3
mAb, and they produce large
amounts of IL-4, IL-10, and IFN- upon primary in vitro stimulation
with anti-CD3 mAb, PMA, and calcium ionophore, or
anti-NKR-P1 mAb (12, 13, 14).
NK1.1+CD4+ T cells have been reported to
augment the IgG response to GPI-anchored Ags (15), and to
down-regulate autoimmune and alloimmune diseases such as diabetes
(16, 17, 18) and acute graft-versus-host disease (GVHD)
(19) via IL-4 and/or IL-10. The selective reduction of
V14-J281 NK1.1+ T cells has
been reported to be associated with the onset of clinical lupus in a
variety of lupus-prone strains of mice (20). The selective
reduction of V24-JQ T cells, a human
homologue of V14-J281 NK1.1+
T cells, and a skewed Th1-like cytokine pattern of the T cells have
been associated with human systemic sclerosis and diabetes (18, 21).
In normal mice, NK1.1+ T cells appear to be preferentially
distributed in different organs, accounting for 20–30% of T cells in
liver and bone marrow and for 0.5–1% of T cells in thymus and spleen.
NK1.1+ T cells are rare in lymph nodes and virtually absent
among gut intraepithelial lymphocytes (1). A previous
report showed that <30% of NK1.1+ T cells in bone marrow
but >80% of them in thymus expressed the invariant
V14-J281 -chain as determined by
quantitative PCR and inverse PCR (22). The developmental
relationships among NK1.1+ T cells in thymus, liver, and
bone marrow is still controversial. The bone marrow T cells expressing
the V14-J281 rearrangement have been
reported to be extrathymically derived from precursors in the bone
marrow itself (23, 24). The bone marrow recently has been
shown to play the crucial role in restoring the homeostasis of
NK1.1+ T cells in liver and spleen after peripheral
depletion (25).
In the current study, we isolated NK1.1+ T cell subsets
from the bone marrow and thymus of wild-type, CD1-/-,
ß2m-/-,
J281-/- and nu/nu C57BL/6
mice and examined their cytokine secretion profiles. Our results
indicate that, consistent with the previous reports (1, 2, 7, 8, 9), almost all NK1.1+ T cells in the thymus
express the invariant V14-J281 TCR and
recognize the ß2m-dependent form of CD1, but they do not
express CD8. However, NK1.1+ T cells in bone marrow include
CD8+ (both CD8+/ß+ and
CD8+/ß-), CD4+, and
CD4-CD8- subsets, and the majority do not
express the invariant V14-J281 TCR.
Nevertheless, most marrow NK1.1+ T cells are CD1 dependent,
but the CD4-CD8- subset is ß2m
independent. Bone marrow NK1.1+ T cells expressing the
invariant V14-J281 TCR in the
CD4+ and CD4-CD8- subsets are the
major source of IL-4 production by these subsets. The CD4+
and CD4-CD8-NK1.1+ T cells that
do not express the invariant V14-J281 TCR
have a Th1-like cytokine profile.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 wild-type mice were obtained from the breeding facility
of the Department of Comparative Medicine at Stanford University.
C57BL/6 ß2m-/- mice
were purchased from The Jackson Laboratory (Bar Harbor, ME). C57BL/6
nu/nu mice were purchased from Taconic Farms (Germantown,
NY). The establishment of
J281-/- founder mice
has been reported previously (26), and those used in the
current study were backcrossed nine generations with C57BL/6 mice.
CD1-/- founder mice with both CD1d1 and CD1d2
genes deleted were established by S. Balk and S. Snapper (Harvard
Medical School, Boston, MA), and those used in the current study were
backcrossed five generations with C57BL/6 mice. Littermate
CD1+/+ mice were used as controls. All the mice
were used at 8–10 wk of age.
mAbs, immunofluorescent staining, and flow cytometric analysis
Single-cell suspensions of thymocytes, obtained from thymus
only, or bone marrow cells obtained from the femur and tibia were
prepared and stained with mAbs as described previously
(27, 28, 29). In most experiments, bone marrow T cells were
enriched using immunomagnetic beads (see below) before staining.
Stainings were performed in the presence of anti-CD16/32 (2.4G2,
PharMingen, San Diego, CA) at saturation to block FcRII/III
receptors; propidium iodide (Sigma, St. Louis, MO) was added to
staining reagents to exclude dead cells. Erythrocytes were excluded by
light scatter gating. Three-color FACS analysis was performed using a
modified dual-laser FACS Vantage (Becton Dickinson, Mountain View, CA),
and data were analyzed using FACS/Desk software (28, 29).
The following conjugated Abs were used for staining:
FITC-anti-CD8 (CT-CD8) and PE-anti-CD8ß (CT-CD8ß)
purchased from Caltag (South San Francisco, CA); Alexia-anti-CD8
(CT-CD8), a gift from D. L. Herzenberg (Stanford University
School of Medicine, Stanford, CA); and
allophycocyanin-anti-TCRß (H57-597), PE-anti-NK1.1
(PK136), and FITC-anti-CD4 (RM4-5) purchased from PharMingen.
Immunomagnetic bead enrichment and sorting of bone marrow NK1.1+ T cells
Sorted CD4+ and CD8+ (collected as a pool and referred to as CD4+/CD8+), CD4+, CD8+, and CD4-CD8-NK1.1+ T cells were obtained from the marrow by flow cytometry after enrichment of bone marrow T cells on immunomagnetic bead columns (Miltenyi Biotec, Auburn, CA). Marrow cells were first incubated with biotinylated anti-Thy-1 (5a-8) mAb (Caltag), then incubated with streptavidin magnetic beads. Thy-1+ cells were positively selected by retention on the magnetic columns and subsequently released. Sorting was performed using a modified dual-laser FACS Vantage (Becton Dickinson) flow cytometer (details of the sorting procedures have been described previously) (27).
In vitro secretion and measurement of cytokines
Sorted NK1.1+ T cells (1 x
105) from bone marrow of wild-type and
gene-deficient mice were stimulated in vitro with 20 ng/ml PMA (Sigma)
and 1 µM ionomycin (Calbiochem, San Diego, CA) in 10% fetal bovine
serum and RPMI 1640 complete medium in 96-well round bottom plates;
they were harvested at the peak time point (48 h) as described
previously (27, 28). Supernatants were assayed in
duplicate for the concentration of IFN-, IL-4, and IL-10 using
commercial ELISA kits (BioSource International, Camarillo, CA).
Assays were developed with avidin-peroxidase and substrate, and plates
were read at 450 nm using a microplate reader (27, 28).
Analysis of V14-J281 RNA expression
by RT-PCR
Total RNA was extracted from unfractionated thymus cells and
from sorted bone marrow
CD4-CD8-NK1.1+
T cells from wild-type and
ß2m-/- mice with the
TRIzol reagent (Life Technologies, Grand Island, NY). For RNA
isolation, 2–3 x 104 cells were used. RNA
was then reverse-transcribed using random hexamer primers followed by
PCR amplification. Optimal conditions for PCR amplification of
ß-actin message, to be used as an internal standard, were established
using titration of the number of amplification cycles and serial
dilution of the cDNA template using primers specific for ß-actin,
followed by densitometry analysis to measure ethidium bromide
luminescence of the PCR products. The number of amplification cycles to
be used for semiquantitative analysis was determined by plotting the
log of the amount of PCR product (measured as absorbance units by
densitometry analysis) as a function of cycle number, to establish a
standard curve. The number of amplification cycles was derived from the
linear portion of the standard curve. Thus, for ß-actin, the cDNAs
underwent 24 cycles of amplification at 94°C for 40 s, 55°C
for 40 s, and 72°C for 40 s, with primers specific for
ß-actin. The sequences of the primers used for ß-actin were
5'-TGGGTCAGAAGGACTCCTATG-3' for the forward primer and
5'-ACCAGACAGCACTGTGTTGGC-3' for the reverse primer. Conditions for
similar semiquantitative PCR analysis for detection of
V14-J281 transcripts
were established as described previously (7). In brief,
the cDNAs underwent 40 cycles of amplification at 94°C for 45 s,
64°C for 15 s, and 72°C for 45 s. The sequences of the
primers used were 5'-TAAGCACAGCACGTGCACAT-3' for
V14, and 5'-CAATCAGCTGAGTCCCAGCT-3' for
J281. The PCR products were resolved on a 3%
agarose gel, and the amount of amplified product was quantified by
densitometry. The levels of
V14-J281 message were
normalized relative to the amount of the ß-actin signal.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NK1.1+ T cells in the thymus of wild-type,
CD1-/-,
ß2m-/-, and
J281-/- C57BL/6 mice
were compared by immunofluorescent staining and two-color flow
cytometric analysis for the NK1.1 vs TCRß, CD4, and CD8 markers as
shown in Fig. 1
. About 0.6% of the
thymus cells from wild-type mice were NK1.1+
ß+ T cells (Fig. 1A), and they
were about equally distributed between CD4+ and
CD4-CD8- cells (Fig. 1, B and C).
CD8+NK1.1+ T cells were not
detected above background staining (Fig. 1D). The
percentages of total NK1.1+ T cells,
CD4+NK1.1+ cells, and
CD4-CD8-NK1.1+
cells in the thymus of CD1-/- (Fig. 1, E–G),
ß2m-/- (Fig. 1, I–K), and
J281-/- (Fig. 1, M–O) mice were reduced about 7- to 10-fold
compared with those in wild-type mice (Fig. 1, A—C). The reduced percentages of
NK1.1+ T cells in the thymus were reflected in
the reduced mean absolute numbers which were 83–88% below the levels
in the wild-type mice (Table I). The
staining patterns of thymus NK1.1+ T cells in
wild-type and littermate control CD1+/+ C57BL/6
mice were similar (data not shown).
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Analyses of NK1.1+ T cell subsets in the
bone marrow of wild-type and gene-deficient mice were performed as
described above. As shown in Fig. 2A, about 2% of whole bone
marrow cells in the wild type stained brightly for TCRß, and about
30% of TCRß+ cells were
NK1.1+. T cells were enriched 10- to 20-fold
using anti-Thy1.2 mAb and immunomagnetic beads to facilitate
further T cell subset analyses. The enrichment procedure did not
significantly change the percentage of NK1.1+ T
cells among TCRß+ T cells (
30%; Fig. 2B), and the yield of NK1.1+ T cells
per mouse was 75–85% after enrichment (data not shown). Compared with
the wild-type marrow, the percentage of NK1.1+ T
cells in whole or enriched CD1-/- bone marrow
(Fig. 2, E and F) was reduced about 4-fold; this
was reflected in a 76% decrease in absolute numbers (Table I
). Thus,
the NK1.1+ T cells in the marrow include a
predominant CD1-dependent subset, and a minority CD1-independent
subset. Although the CD1-/- mice were
backcrossed on the C57BL/6 background for five generations, it is
possible that genes other than that encoding CD1 contributed to the
reduction of the NK1.1+ T cells in the marrow.
Accordingly, we analyzed the marrow from CD1+/+
littermates (Fig. 2, C and D) of the
CD1-/- mice and found that the percentage of
NK1.1+ T cells was similar to that of wild-type
C57BL/6 mice. The percentage of NK1.1+ T cells in
the enriched ß2m-/-
marrow (Fig. 2G) was reduced by about 50%, but this
reduction was not detected in the whole bone marrow. This discrepancy
may be due to the improved resolution and quantitation of subsets in
the enriched marrow. The absolute number of
NK1.1+ T cells in the
ß2m-/- marrow was
reduced by 53% (p < 0.01, two-tail Student’s
t test; Table I). The percentages of
NK1.1+ T cells among all marrow T cells in
the ß2m-/- and
wild-type mice (Fig. 2, B and H) remained about
the same due to a reduction in the absolute numbers of
CD8+ NK1.1- T cells (data
not shown). The percentage of NK1.1+ T cells in
J281-/- marrow (Fig. 2, I and J) was similar to that in the wild-type
marrow (Fig. 2, A and B), and the absolute
numbers were not significantly different (Table I). The percentage of
NK1.1+ T cells in age-matched (8–10 wk)
nu/nu marrow (Fig. 2, K and L) was
reduced 50- to 100-fold to the level of background staining.
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ß+ T cells in the enriched marrow
cells of wild-type mice (Fig. 3A, enclosed in box) were
analyzed for CD4 and CD8 markers together, and CD4 and CD8 markers
separately (Fig. 3, B–D). Three discrete
populations of NK1.1+ T cell subsets including
CD4-CD8-,
CD4+, and CD8+ were
observed. The percentage of the CD8+ subset among
T cells was about twice that of the CD4+ subset
and was similar to that of the
CD4-CD8- subset. This was
reflected in the absolute numbers of the NK1.1+ T
cell subsets harvested from marrow (Table I). The ratio of all
CD8+ vs CD4+ cells among
all TCRß+ T cells in the wild-type marrow
was about 2:1 using the absolute numbers (Table I). In additional
experiments, gated TCRß+
NK1.1+ cells (Fig. 4A) from the wild-type
enriched marrow were analyzed for CD8 vs CD8ß markers (Fig. 4B). About 70% of the
CD8+NK1.1+ T cells stained
brightly for both markers (Fig. 4B, enclosed in upper
box), and about 30% stained dully for only the CD8 marker
(Fig. 4B, enclosed in lower box). Staining for
CD8 markers was also performed on gated NK1.1+
TCRß+ cells from
ß2m-/- bone marrow and
on gated NK1.1- TCRß+
cells from wild-type spleen (Fig. 4, C and E,
respectively). However, the cells did not express levels of either
marker above background controls in the case of the
ß2m-/- marrow cells
(Fig. 4D). In the case of the wild-type spleen, almost all
CD8+ cells expressed both the CD8 and CD8ß
markers, and few, if any, expressed only the CD8 marker (Fig. 4F).
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, E–H) compared with wild type (Fig. 3, A–D), and their absolute numbers were reduced by
72–80% (p < 0.001, two-tail Student’s
t test; Table I). Surprisingly, the percentage of
CD4-CD8-NK1.1+
T cells in ß2m-/-
marrow (Fig. 3J) was increased compared with wild type, and
the absolute numbers were similar (Table I). In contrast, there was a
marked depletion of
CD8+NK1.1+ T cells in
ß2m-/- mice (Fig. 3L), and the reduction of the absolute number of
CD8+ NK1.1+ T cells was
98% (p < 0.001, two-tail Student’s
t test, Table I). The percentage of
CD4+ NK1.1+ T cells in
ß2m-/- marrow was
reduced (Fig. 3K) compared with the wild type, and the
absolute numbers were reduced by about 3-fold
(p < 0.01, two-tail Student’s t
test, Table I). The percentage (Fig. 3N) and the absolute
number of
CD4-CD8-NK1.1+
T cells in J281-/-
mice were not significantly reduced (p > 0.05,
two-tail Student’s t test; Table I) compared with those in
wild-type mice. The percentage (Fig. 3P) and absolute number
of CD8+NK1.1+ T cells were
significantly increased by 38% (p < 0.05,
two-tail Student’s t test; Table I). On the other hand, the
percentage of CD4+NK1.1+ T
cells in J281-/- mice
was reduced about 2-fold (Fig. 3O), and the absolute number
was reduced by 43% (p < 0.05, two-tail
Student’s t test; Table I). Cytokine secretion patterns of NK1.1+ T cell subsets in marrow
It has been reported that CD4+ and
CD4-CD8-NK1.1+
T cells from thymus and peripheral tissues such as spleen and liver
produced large amounts of IFN-, IL-4, and IL-10 in response to in
vitro stimulation with anti-CD3 mAb and PMA plus ionomycin
(1, 12, 14, 30). Because some subsets of
NK1.1+ T cells in the marrow were not identified
in the thymus, the cytokine secretion profiles of the
NK1.1+ T cell subsets in the bone marrow of
wild-type and gene-deficient C57BL/6 mice were studied. Sorted
CD4+/CD8+
(CD4+ and CD8+ T cells as a
pool), CD4+, CD8+, and
CD4-CD8-NK1.1+
T cells were stimulated with PMA and ionomycin for 48 h, and the
culture supernatants were assayed for concentrations of IL-4, IL-10,
and IFN-. As shown in Table II,
control sorted
CD4+/CD8+NK1.1-
T cells from the peripheral blood of wild-type mice produced large
amounts of IFN-, but little IL-4 or IL-10, and the ratio of IFN-
to IL-4 was 56:1. In contrast, the sorted
CD4+/CD8+ and
CD4-CD8-NK1.1+
T cells from the marrow produced large amounts of IFN-, IL-4, and
IL-10. The ratio of IFN- to IL-4 was 1.1:1 for
CD4+/CD8+ and 1.9:1 for
CD4-CD8-NK1.1+ T cells. It is of
interest that the sorted
CD8+NK1.1+ T cells were
similar to the sorted
CD4+NK1.1+ T cells and also
produced large amounts of IFN-, IL-4, and IL-10 (Table II).
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281-/- mice was
different from that of wild-type mice. Although the
CD4-CD8-NK1.1+
T cells from the marrow of both
ß2m-/- and
J281-/- mice produced
comparable amounts of IFN- compared with the
CD4-CD8-NK1.1+
T from wild-type marrow, they produced 10- to 20-fold less IL-4 and
IL-10 (p < 0.001, two-tail Student’s
t test; Table II). The IFN- secretion of the
CD4+/CD8+NK1.1+
T cells from the marrow of
ß2m-/- and
J281-/- mice was also
similar to that of
CD4+/CD8+
NK1.1+ T cells from wild-type marrow, but the
IL-4 production of the
CD4+/CD8+NK1.1+
T cells was reduced 8-fold from
ß2m-/- marrow, and
3-fold from J281-/-
marrow. In the case of
ß2m-/- marrow, the
CD4+ cells were the source of cytokines of the
sorted
CD4+/CD8+NK1.1+
T cells, because CD8+ cells were not easily
detected. Thus, changes in the cytokine profile are due to changes on
the CD4+NK1.1+ T cells. In
the case of the
J281-/- marrow, the
CD8+cells were about 5-fold more numerous than
CD4+ cells, and the changed cytokine profile
reflects the contribution of both CD4+ and
CD8+NK1.1+ T cells.
Changes in V14-J281 TCR gene
expression in ß2m-/- mice
Because the cytokine profiles were markedly different in the
CD4-CD8-NK1.1+
T cells in marrow from wild-type and
ß2m-/- mice, we
examined changes in the expression of the
V14-J281 TCR
-chain gene in these mice using a semiquantitative RT-PCR assay.
Primers were designed to amplify the invariant TCR -chain cDNA as
well as the ß-actin cDNA. PCR conditions were set so that the
intensity of the amplified cDNA products was on the linear portion of
titration curves comparing band intensity with the PCR cycle number.
RNA was isolated from 2 to 3 x 104 cells
from wild-type and
ß2m-/- mice, and
intensity of the
V14-J281 TCR signal
was normalized relative to the ß-actin signal. Fig. 5 shows the
V14-J281 and
ß-actin amplified products and signal intensities obtained from
wild-type and ß2m-/-
whole thymus cells, and those of sorted
CD4-CD8-NK1.1+
T cells from the bone marrow. Lanes 1 and
2 show a marked reduction in the levels of amplified
V14-J281 cDNA from
wild-type and ß2m-/-
whole thymus cells, respectively, as judged by the intensity of the
bands and densitometry values. This was expected due to the reduced
number of NK1.1+ T cells in the
ß2m-/- thymus.
Lane 3 shows that an amplified
V14-J281 signal was
easily detectable using sorted
CD4-CD8-NK1.1+
T cells from wild-type marrow with an intensity similar to that of
whole thymus. This suggests that only a small percentage of
CD4-CD8-NK1.1+
T cells in the bone marrow express the
V14-J281 gene.
Lane 4 shows that the intensity of this band and the
associated densitometry values were markedly reduced in
ß2m-/- marrow compared
with wild-type marrow, despite equal numbers of sorted
CD4-CD8-NK1.1+
T cells. The intensities of the ß-actin bands in lanes
1–4 were comparable.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/ß T cells that co-express receptors of the NK lineage and have
the unique potential to very rapidly secrete large amounts of cytokines
such as IL-4 (1). Previous studies showed that >85% of
NK1.1+ T cells in thymus express the invariant
V14-J281 TCR and that
this unique TCR recognizes CD1 in association with
ß2m (1, 7, 8, 9). The latter molecule
has been shown to positively select the thymic
NK1.1+ T cells (5). The
CD8+NK1.1+ T cells in
thymus are theorized to be depleted by negative selection
(1). The current study also showed that the thymus of
wild-type mice contained only CD4+ and
CD4-CD8-NK1.1+
subsets. The absolute numbers of CD4+ and
CD4-CD8-NK1.1+
T cells in the thymus of CD1-/-,
ß2m-/-, and
J281-/- mice were
reduced by about 10-fold, as in previous studies (7, 8, 9, 26).
The pattern of NK1.1+ T cell subsets in bone
marrow is different from that in thymus. NK1.1+ T
cells make up about 30% of T cells in bone marrow compared with
0.5–1% in thymus and include CD4+,
CD8+, and
CD4-CD8- subsets. The
CD8+ subset was twice as numerous as the
CD4+ subset and similar in number to the
CD4-CD8- subset. Judging
from flow cytometric analyses, the
CD8+NK1.1+ T cells were
made up of about 70% expressing the CD8ß
(+ß+) heterodimer, and
30% expressing the CD8
(+ß-) homodimer.
CD8 homodimeric T cells are also found among intestine epithelial
lymphocytes and are extrathymically derived (31, 32). The
presence of CD8+NK1.1+ T
cells in the marrow but not in the thymus indicates that those
NK1.1+ T cells are extrathymically derived. It is
also possible that CD8 is an activation marker on
CD4-CD8-NK1.1+
T cells and that it does not reflect the tissue origin of the
CD8+ß-NK1.1+
T cell subset. All NK1.1+ T cell subsets
including the CD8+ subset were markedly depleted
in athymic nu/nu mice. Thus, the bone marrow
CD8+NK1.1+ T cells were
clearly dependent on the presence of the thymus in the 2-mo-old mice
used in the current study. Possible explanations of this apparent
paradox are that the wing helix nude (nu) mutation expressed
in epithelial cells of nu/nu mice (33, 34)
interferes with extrathymic development of the
CD8+NK1.1+ T cells, or that
the thymus facilitates extrathymic development of these cells by
exporting humoral substances or facilitating cells. Alternatively, the
CD8+NK1.1+ T cells in the
marrow may be derived from thymic emigrants that alter their phenotypic
and functional characteristics when they localize to the bone marrow.
Some
V24-JQ+
T cells, the human homologue of murine NK1.1+ T
cells (35), have been reported to be
CD8dim/ß- T cells
(36), and may be homologues of the
CD8+/ß-NK1.1+
T cells (which also had dim staining for CD8) observed in the mouse
marrow. The human
CD8dim/ß- T cells
represented the majority of
V24-JQ+
Vß11+ T cells in some
donors, but in others the majority of the cells were
CD4-CD8-, the phenotype
reported initially for most
V24-JQ+
T cells (35).
Evidence that the bone marrow is a direct source of
NK1.1+ T cells expressing the
V14-J281 TCR includes
the presence of DNA deletion circles specific for this TCR -chain
gene rearrangement, and the expression of RAG-1 and RAG-2 genes in
marrow cells which are not members of the B cell lineage (23, 24). In the current study, the bone marrow of nu/nu
mice older than 3 mo was not investigated for the content of
NK1.1+ T cells. Because T cells slowly develop in
the lymphoid tissues of nu/nu or thymectomized irradiated
mice and are easily detected by 3–6 mo of age (37, 38),
it is possible that NK1.1+ T cells gradually
develop in the marrow of the older mice. Late development may account
for previous reports of the presence of NK1.1+
CD3+ T cells in the lymphoid tissues of such
athymic mice (23, 24, 39) and for the lack of detection in
the current study.
CD8+NK1.1+ T cells were
considerably reduced (by 72%) in the CD1-/-
bone marrow compared with those in the wild-type marrow, indicating
that the predominant subset is CD1 dependent, as previously reported
for NK1.1+ T cells in other tissues. However, a
minority subset of CD1-independent CD8+
NK1.1+ T cells persisted and accounted for about
30% of the wild-type number. In contrast, the absolute number of
CD8+NK1.1+ T cells was
significantly increased in
J281-/- marrow, and
almost completely depleted in
ß2m-/- marrow. These
results indicate that few
CD8+NK1.1+ T cells
express the invariant
V14-J281 TCR, and the
CD8+ NK1.1+ cells still
recognize the ß2m-dependent form of CD1. The
latter molecule presumably is required for positive selection of the
marrow CD8+NK1.1+ T cells,
but it does not negatively select the marrow cells, as proposed for
this subset in the thymus (1). This dichotomy may be due
to differences in tissue specific ligands that bind to CD1 and interact
with predominantly
V14-J281 receptors in
the thymus, and with other -chain receptors in the marrow
(40, 41, 42).
The number of
CD4-CD8-NK1.1+
T cells was not significantly reduced in either
J281-/- or
ß2m-/- bone marrow,
indicating that a substantial fraction of these
CD4-CD8-NK1.1+
cells do not express the invariant
V14-J281 TCR and may
recognize a ß2m-independent form of CD1. The
latter form is likely to have an identical amino acid sequence to that
of the ß2m-dependent form, but the
CD4-CD8-NK1.1+
TCR may interact with different segments of CD1 or with different CD1
ligands, based on differences in conformation engendered by the
ß2m association. An alternative explanation
is that some subsets of marrow
CD4-CD8-NK1.1+
T cells recognize a ß2m-independent molecule
other than CD1, and that these NK1.1+ T cells
expand in ß2m-/- mice.
The presence of a small residual population of
NK1.1+ T cells in the marrow of
CD1-/- mice (Fig. 3) is consistent with this
notion. Ongoing experiments, beyond the scope of the current study,
will test this explanation, including those that will test the
reactivity of sorted marrow
CD4-CD8-NK1.1+
T cells to ß2m-deficient CD1-transfected cells,
and those that will search for marrow
CD4-CD8-NK1.1+
T cells in
ß2m-/CD1-/-
(double gene deficient) mice.
The presence of
V14-J281-
T cells among the CD8+ and
CD4-CD8-NK1.1+
T cells in marrow is consistent with the previous reports that more
than 70% of NK1.1+ T cells in bone marrow do not
express the invariant
V14-J281 TCR
(22). Of the <30% of NK1.1+ T
cells in the marrow that express the invariant TCR, the large majority
are likely to be contained within the CD4+
NK1.1+ T cell subset, and only a small percentage
are within the
CD4-CD8-NK1.1+
T cell subset. The similar PCR signals in the whole thymus (of which
0.6% are NK1.1+ T cells) and in the sorted
marrow
CD4-CD8-NK1.1+
T cells is consistent with the latter distribution. Previous studies of
the V14-J281
expression in marrow NK1.1+ T cells did not
examine the expression within isolated NK1.1+ T
cell subsets (22, 26). In one of these studies
(26), the percentage of NK1.1+ T
cells among all T cells in the marrow of both
J281+/+ and
J281-/- mice was
considerably lower than in the current study. This discrepancy is most
likely due to the use of
J281-/- and control
mice in the present study after nine backcross generations, whereas the
previous study used mice after only three backcross generations
(26). The mixture of NK1.1+ T cells
in the marrow is consistent with two different types of
NK1.1+ T cell hybridomas; some anti-CD1
V14-J281-
T cell hybridomas recognize a ß2m-independent
form of CD1, whereas the
V14-J281+
hybridomas only recognize a ß2m-dependent form
of CD1 (43). A
CD4-CD8-
TCRß+ V4.4
anti-CD1 T cell clone also has been reported to recognize a
ß2m-independent form of CD1 (27, 44). In addition, a ß2m-independent form
of CD1 previously has been reported to be expressed by human intestinal
epithelium cells (45).
Although the
CD4-CD8-NK1.1+
T cells were abundant in the
J281-/- and
ß2m-/- marrow, they
were not detected in the thymi of these mice. Studies of the
cytokine secretion profile of the
CD4-CD8-NK1.1+
T cells in the marrow of the wild-type and gene-deficient mice showed
that a marked change occurred in the latter mice. Whereas the
CD4-CD8-NK1.1+
T cells in the wild-type mice secreted high levels of IFN-, IL-10,
and IL-4, those in the
J281-/- and
ß2m-/- mice secreted
high levels of IFN- but about 10-fold reduced levels of IL-10 and
IL-4 (Th1-like pattern). This suggests that marrow
CD4-CD8-NK1.1+
T cells expressing the
V14-J281 TCR and
recognizing the ß2m-dependent form of CD1
secrete high levels of all three cytokines, but that the
V14-J281-
CD4-CD8-NK1.1+
T cells that recognize a ß2m-independent ligand
secrete a Th1-like pattern. Both types of NK1.1+
T cells are present among
CD4-CD8-NK1.1+
T cells in the marrow of wild-type mice, as judged by the expression of
this TCR -chain gene by PCR analysis of the sorted
CD4-CD8- subset in the
latter mice. The reduced PCR signal in the sorted cells from
ß2m-/- mice showed that
few, if any, of the residual cells express this TCR. The cytokine
pattern of the
V14-J281-
subset is only revealed in the
ß2m-/- and
J281-/- mice, and the
cytokine pattern of the
V14-J281+
subset is dominant in the wild-type mice.
The CD4+NK1.1+ T cells in
the marrow are also made up of a combination of
V14-J281+
and
V14-J281-
subsets, based on the results using
ß2m-/- and
J281-/- mice. The
reduction of the absolute number of
CD4+NK1.1+ T cells by 73%
and 43%, respectively, in these gene-deficient mice supports this
notion. In addition, the cytokine pattern of the residual
CD4+ NK1.1+ T cells in
ß2m-/- mice showed a
Th1-like pattern similar to that of the
CD4-CD8-NK1.1+
T cells in the same mice. Thus, the Th1-like pattern appears to be
secreted by both
V14-J281-
CD4+ and
V14-J281-
CD4-CD8-NK1.1+
T cells, that recognize a ß2m-independent
ligand. Because there was a residue of NK1.1+ T
cells in the CD1-/- mice, some fraction of the
NK1.1+ T cells may use molecules other than CD1
for positive selection.
After the submission of our manuscript, a report by Eberl et al. (46) was published which also showed that CD8+NK1.1+ T cells were more numerous than CD4+NK1.1+ T cells in bone marrow, despite the limited detection of CD8+NK1.1+ T cells in the thymus. However, the marrow CD8+NK1.1+ T cells were reported to be CD1 independent, whereas the current report found both CD1-independent and CD1-dependent CD8+NK1.1+ T cell subsets. Explanations of the different findings include the use of nylon wool columns for marrow cell enrichment (46) that may have depleted the CD1-dependent subset, and the use of CD1-/- mice backcrossed to the C57BL/6 background for three generations compared with those backcrossed for five generations in the current report. Flow cytometric patterns showing analyses of the CD8+NK1.1+ T cells in the marrow of CD1-/- mice or in their littermate controls were not shown by Eberl et al. (46), so that a direct comparison with patterns in the current report cannot be made.
In conclusion, almost all the NK1.1+ T cells in
the thymus express the
V14-J281 TCR, whereas
the bone marrow NK1.1+ T cells are more
heterogeneous and have a different balance of both
V14-J281+
and
V14-J281-
NK1.1+ T cells, each of which expresses different
cytokine profiles and different accessory molecules, such as CD8. The
marrow
V14-J281-
NK1.1+ T cells appear to arise extrathymically,
in view of their paucity in the thymus and their unusual phenotypes
(i.e., CD8+ß-), but
the
V14-J281+
NK1.1+ T cells are likely to arise from both
thymic and extrathymic sources. Recent studies show that the
NK1.1+ T cells in the marrow suppress GVHD
induced by NK1.1- T cells, and that this
NK1.1+ T cell function, especially in the
CD4-CD8- subset, is
mediated by IL-4 (19). The current study indicates that
the suppressive activity in these
CD4-CD8-NK1.1+
T cells is confined to the
V14-J281+
cells, because the latter are the key source of IL-4. The selective
loss of
V14-J281+
NK1.1+ T cells in lupus-prone mouse strains
(20) may result in a shifted pattern of cytokine secretion
(Th1-like) in the residual CD1-recognizing T cells. CD1-recognizing T
cells with a Th1-like pattern have been reported to induce lupus
(27). Thus, the balance of
V14-J281+
and
V14-J281-
NK1.1+ T cells described herein may influence
autoimmune and alloimmune diseases.
|
Acknowledgments |
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Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Samuel Strober, Division of Immunology and Rheumatology, Stanford University School of Medicine,300 Pasteur Drive, Room S105B, Stanford, CA 94305. E-mail address:
3 Abbreviations used in this paper: ß2-m, ß2-microglobulin; GVHD, graft-versus-host disease.
Received for publication April 13, 1999. Accepted for publication September 7, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/ß-T cell receptor (TCR)+CD4-CD8- (NK T) thymocytes prevent insulin-dependent diabetes mellitus in nonobese diabetic (NOD)/Lt mice by the influence of interleukin (IL)-4 and/or IL-10. J. Exp. Med. 187:1047.14-J281 transgenic nonobese diabetic mice against diabetes. J. Exp. Med. 188:1831.24JQ T cells in type 1 diabetes. Nature 391:177.[Medline]
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24JQ antigen receptor in patients with systemic sclerosis. J. Exp. Med. 182:1163.14+ TCR -chain in NK1.1+ T cell populations. Int. Immunol. 7:1157.14+ T cells. J. Exp. Med. 177:1399.14J281 TCR. Int. Rev. Immunol. 11:31.[Medline]
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14 NKT cells in IL-12-mediated rejection of tumors. Science 278:1623./ß T cells demonstrates preferential use of several Vß genes and an invariant TCR -chain. J. Exp. Med. 178:1.24 and Vß11 coexpression defines a human NK1 T cell analog containing a unique Th0 subpopulation. J. Immunol. 159:5862.[Abstract]
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