By
From the * Institut National de la Santé et de la Recherche Medicale U267, Hôpital Paul Brousse,
Villejuif 94807, France; and the Institut National de la Santé et de la Recherche Medicale U429,
Hôpital Necker, Paris 75743, France
In this report, we have assessed the lineage relationships and cytokine dependency of natural
killer (NK) T cells compared with mainstream TCR- T cells and NK cells. For this purpose, we studied common
chain (
c)-deficient mice, which demonstrate a selective defect in
CD3
NK cell development relative to conventional TCR-
T cells. NK thymocytes differentiate in
c
mice as shown by the normal percentage of TCR V
8+ CD4
CD8
cells and
the normal quantity of thymic V
14-J
281 mRNA that characterize the NK T repertoire.
However,
c-deficient NK thymocytes fail to coexpress the NK-associated markers NKR-P1
or Ly49, yet retain characteristic expression of the cytokine receptors interleukin (IL)-7R
and
IL-2R
. Despite these phenotypic abnormalities,
c
NK thymocytes could produce normal
amounts of IL-4. These results define a maturational progression of NK thymocyte differentiation where intrathymic selection and IL-4-producing capacity can be clearly dissociated from
the acquisition of the NK phenotype. Moreover, these data suggest a closer ontogenic relationship of NK T cells to TCR-
T cells than to NK cells with respect to cytokine dependency.
We also failed to detect peripheral NK T cells in these mice, demonstrating that
c-dependent
interactions are required for export and/or survival of NK T cells from the thymus. These results suggest a stepwise pattern of differentiation for thymically derived NK T cells: primary selection via their invariant TCR to confer the IL-4-producing phenotype, followed by acquisition of NK-associated markers and maturation/export to the periphery.
NK T cells are a specialized subset of T cells that share
surface markers with the NK cells and have unique
properties with respect to their TCR diversity and specificity, as well as their ultimate biological functions (1). NK
T cells comprise both CD4 The potential to produce IL-4 and the NK phenotype
are two characteristic properties of NK T cells that are
likely acquired during their selection by CD1 at an early
ontogenic stage (15). This hypothesis has been strengthened by recent studies of V A separate question in the development of NK T cells
involves the role of cytokines. The common Mice.
Mice deficient for the common cytokine receptor Cell Preparation and FACS® Analysis.
Thymocyte and splenocyte (red cell-depleted) suspensions were prepared aseptically
in HBSS after pressing through sterile mesh filters. Liver lymphocytes were isolated using discontinuous Percoll gradients (20)
with minor modifications. Cells were stained using combinations
of directly conjugated mAbs: FITC-anti-TCR- Quantitative RT-PCR.
Total RNA was extracted with acid-
guanidinium (22) and ethanol-precipitated with the addition of 5 µg
of glycogen before resuspension in 20 µl of DEPC water. Reverse
transcription and quantitative PCR amplification were carried out
as previously described (23) using oligonucleotides specific for C In Vivo and In Vitro IL-4 Production.
Administration of purified anti-CD3 (clone 145-2C11; 2 µg i.v.) and subsequent in vitro culture of splenocyte suspensions for IL-4 production were
performed exactly as described (13). To evaluate cytokine production following stimulation in vitro, CD8 A semiquantitative PCR approach (23) was used to enumerate NK T cells
by exploiting the fact that these cells exhibit a restricted
TCR-
The presence of
NK T thymocytes express a unique constellation of cell surface markers, including intermediate density TCR-
These results, together with the V Because NK T cells are selected to a similar degree in the
thymus of The unique ability of NK T cells to produce IL-4 after TCR triggering has
been one main characteristic of this lymphoid subset (1, 13,
14). When CD8 Table 1.
IL-4 Production from NK T Cells: Production of
IL-4 from In Vitro-stimulated Thymocytes
CD8
(double negative, DN)
and CD4+ TCR-
bearing T cells (4, 5), which coexpress a cluster of NK cell markers, including receptors of
the C-lectin Ly49 and NKR-P1 (including the NK1.1 antigen) families (1, 6, 7). NK T cells express high levels of
the IL-2R
molecule, a shared cytokine receptor chain
used by IL-2 and IL-15, which is also found on NK cells
and TCR-
T cells, but at low levels on conventional TCR-
T cells (8). Moreover, although the level of TCR
expression on conventional T cells is high, NK T cells express intermediate TCR levels (TCR-
int). The TCR
repertoire of NK T cells is markedly restricted: TCR-
chain usage includes V
8, V
7, and V
2, whereas the
TCR-
chain is mostly an invariant
chain using the
V
14 and J
281 segments with a conserved junctional sequence (9, 10). The limited TCR-
diversity of NK T
cells suggested that these cells interact with a similarly nonpolymorphic ligand (4, 10). Studies using T cell hybridomas derived from NK T cells have clearly identified the nonpolymorphic MHC class Ib CD1 molecule as the ligand
recognized by these peculiar TCR (11). NK T cells are remarkable for their ability to produce large amounts of cytokines after TCR stimulation (12), notably IL-4 (12, 13).
This prompt IL-4 production by NK T cells has suggested
a model in which these cells are one of the major determining factors influencing the final TH1/TH2 profile of
immune responses (13, 14).
14-J
281 transgenic mice (16),
which have increased numbers of NK T cells, increased IL-4
production and augmented baseline levels of IgE and IgG1
(16). Still, a role for the NK-associated molecules during the
selection of NK T cells has not been excluded, and a coreceptor function for the NK1.1 molecule has been suggested (2) based on the presence of the amino acid Cys-X-Cys-
Pro motif in the NK1.1 cytoplasmic domain (17). This motif
was first identified in the CD4 and CD8-
coreceptors as
the region that specifically interacts with p56lck, a tyrosine
kinase whose association with the CD4 or CD8 coreceptors is important for optimal T cell activation (18).
chain (
c)1,
is a critical component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15.
c-deficient mice have abnormal lymphoid development, with a complete absence of NK cells,
TCR-
T cells, and gut-associated intraepithelial lymphocytes (19). In contrast, TCR-
T cells and B cells are
present, albeit in reduced numbers. Therefore,
c
mice represent a useful system to assess lineage relationships between
various lymphoid subpopulations. In this report, we have studied NK T cell development in
c
mice. The presence
of NK thymocytes in
c
mice suggest a closer ontogenic
relationship of NK T cells to mainstream TCR-
T cells
than to NK cells with respect to cytokine dependency.
Based on our phenotypic and functional analyses of
c
NK thymocytes, we propose and discuss a stepwise model
of NK T cell differentiation.
chain,
c (initially identified as the IL-2 receptor
c, reference
19), were maintained in our conventional animal facility and were
of a mixed background (129/Ola/BALB/c or 129/Ola/BL/6).
For the analysis of NK-associated markers including NK1.1 and
Ly49 members, female mice heterozygous for the X-linked
c
mutation (from the fourth backcross to BL/6 with confirmed
NK1.1 and Ly49 expression) were mated to normal BL/6 males
and the subsequent
c+ or
c
male mice were analyzed for
NK1.1 expression. Mice were analyzed between 4 and 10 wk of age.
(clone H57),
biotin-anti-heat-stable antigen (HSA, clone J11d), and biotin-
anti-V
8 (clone F23.1); FITC-anti-IL-7R
chain (21) (purified
and locally conjugated by standard methods), PE-anti-HSA, biotin-anti-TCR-
, FITC-anti-IL-2R
, FITC-anti-Ly49C, and
PE-anti-NK1.1 (all from PharMingen, San Diego, CA); PE-antiCD4, FITC-anti-CD8, and Tricolor-streptavidin (Caltag Laboratories, San Francisco, CA). FITC-anti-Ly49A (JR9-319) was the
gift of J. Roland (Institute Pasteur, Paris, France). Three-color immunofluorescence analysis was performed using a FACScan® flow
cytometer (Becton Dickinson & Co., Mountain View, CA) and analyzed using CellQuest software.
,
V
14, and J
281 (10). It should be stressed that because there is no allelic exclusion for the
chain locus of the TCR (24), only
enrichment for a certain VJ combination in a peculiar sample can
be detected by PCR analysis using PCR primers specific for V
and J
segments. If the amount of starting material is high enough
(more than ~2 × 104 cell equivalents), in samples that do not
contain V
14-J
281 invariant
chains (such as those from
2microglobulin [
2m]
/
mice), there is always a background signal
related to the amplification of nonselected out of frame and/or
polymorphic TCR-
chains using the same VJ combination. Indeed, polyclonal sequencing of such V
14-J
281 PCR products
demonstrated their polymorphism (reference 10; data not shown).
In the kinetic PCR method we are using, if one considers two
samples containing the same amount of C
, a shift of n cycles along
the x axis of the two amplification curves represents an ~1.8nfold difference in V
14-J
281 expression.
thymocytes were purified after a one-step killing with anti-CD8 mAb (TiB-211; American Type Culture Collection, Rockville, MD) plus low-toxic M
rabbit complement (Cederlane Laboratories, Hornby, Canada).
Viable cells were recovered by centrifugation over a density gradient. CD8
thymocytes (3 × 105) were cultured in RPMI-1640
medium, 10% FBS, 50 µM 2-mercaptoethanol, 2 mM glutamine
with 3 × 104 antigen-presenting cells and soluble anti-CD3 at 5 µg/ml in a total volume of 0.4 ml for 48 h, with or without exogenous cytokines (thymic stromal cell-derived lymphopoietin,
TSLP [25], at 10 ng/ml). Supernatants were harvested and IL-4
content measured using the CT.4S cell line. Responses were
compared with those elicited by known amounts of murine IL-4.
NK Thymocytes Develop in the Absence of c.
chain repertoire, using the V
14 segment joined to J
281 (10). We quantitated the amounts of V
14-J
281
TCR-
chain in CD8
(CD4
CD8
[DN] and CD4+
single-positive [SP]) thymocytes from
c+,
c
, and
2m
/
mice.
2m
/
cells were used as control as it has been previously shown that NK T cells require the
2m-associated
CD1 molecules in order to be selected efficiently (4, 5, 10,
11, 15). As shown in Fig. 1, C
transcripts were found
equally in all three cDNA preparations. The amount of
V
14-J
281 mRNA was similar in both
c+ and
c
thymi (within threefold) and largely increased relative to
2m
/
thymi, which lack NK T thymocytes. Direct polyclonal sequencing the V
14-J
281 amplicons from
c+
and
c
thymi verified the presence of the canonical
CDR3 motif, whereas
2m
/
amplicons were polymorphic (data not shown).
Fig. 1.
V14-J
281 invariant
chain is normally expressed in mature thymocytes of
c
mice. Duplicates samples of 5 × 105 CD8
thymocytes were obtained from the indicated mice and RNA extracted. After reverse transcription, the indicated genes were amplified and the
amount of amplicons quantified at the indicated cycle. Averaged duplicate
values are shown. Representative of three independent experiments.
[View Larger Version of this Image (0K GIF file)]
c
NK T thymocytes was confirmed
by analysis of DN thymocytes for the expression of V
8
(Fig. 2). The TCR-
repertoire of DN NK T cells is
highly restricted, with ~50% of cells using V
8 (4, 7, 10).
DN thymocytes from both
c+ and
c
mice contained a
population of V
8+ cells (~5%), which was not detected
in DN thymocytes from
2m
/
mice. Percentages of V
8+
cells amongst mature (HSAlo) DN thymocytes were also
comparable between
c+ (30.3 ± 7.8%) and
c
(20.7 ± 4.7%) mice (data not shown). Taken together, these results suggest that NK T cells are found at the same relative frequency in
c
thymi as in
c+ thymi, although their absolute numbers are reduced by 20-fold in parallel with the
overall decrease in thymopoiesis seen in
c
mice (19).
These results suggest that generation of NK T cells after interactions with CD1 molecules can proceed in the absence of
c. In this way, selection of NK T cells parallels that of the conventional CD4+ and CD8+ TCR-
T cells, which
can be generated independent of
c (19, 26; DiSanto, J.P.,
unpublished data). The ability of NK thymocytes to develop
in the absence of
c clearly distinguishes this lymphoid subset from classical NK cells, which have an absolute requirement for
c-dependent interactions in their development (19).
Fig. 2.
c
thymocytes contain normal proportions of DN TCR-
V
8-expressing cells. Thymocytes from
c+,
c
, and
2m
/
mice
were stained with CD8-FITC, CD4-PE, and F23.1-biotin followed by
Tricolor streptavidin. Histograms show V
8 expression on gated DN
(CD4
CD8
) thymocytes.
[View Larger Version of this Image (0K GIF file)]
c
Mice.
(TCR-
int) and coexpression of NK-associated antigens, including members of the NKR-P1 and Ly49
families (1). Next, we investigated whether NK T thymocytes from
c
mice maintained this particular phenotype using mice on the C57BL/6 background. Mature thymocytes (expressing low levels of heat-stable antigen, HSAlo)
were examined for a variety of markers in combination with
TCR-
chain expression. Thymi from BL/6
c+ mice contained a subpopulation of TCR-
int cells expressing the
NK1.1 marker (Fig. 3). In
c+ mice, a fraction of the
NK1.1+ thymocytes coexpressed Ly49C or Ly49A, and all
cells were positive for the IL-2R
and IL-7R
chains (Fig.
3; data not shown). In contrast, no NK1.1+ or Ly49C+
cells were found in thymi from BL/6
c
mice, although
TCR-
int cells were clearly detectable (Fig. 3). In
c
mice, these TCR-
int cells expressed high levels of IL-7R
(like their
c+ counterparts) and somewhat reduced levels
of IL-2R
(Fig. 3).
Fig. 3.
Phenotype of NK thymocytes. Dot plots show expression of
NK1.1, Ly49C, IL-7R or IL-2
as a function of TCR-
chain expression on mature (HSAlo) thymocytes from
c+ or
c
BL/6 mice. Boxed
regions indicate the NK thymocytes that have characteristic TCR-
int expression.
[View Larger Version of this Image (0K GIF file)]
14-J
281-specific PCR
data and expression of V
8 on DN thymocytes, demonstrate that NK T cells are present in the thymus of
c
mice,
although they do not express the NK-associated markers. This suggests that the generation and selection of NK thymocytes can be dissociated from the acquisition of the
NKR-P1 and Ly49 markers, and that the NK phenotype is
a contingent phenomenon, which alone cannot be used to
define a particular lineage. Concerning the potential function of NKR-P1 molecules as coreceptors for the recognition of CD1 during selection of NK T cells (2), our results
show that NKR-P1 expression is not strictly required for
positive selection of NK T cells on CD1, although we cannot rule out that additional interactions are afforded to the
selection process by NKR-P1 molecules. The coabsence of
Ly49 family molecules on
c
NK thymocytes is consistent
with the NK-associated markers being encoded by their
genetically linked loci or the NK gene complex (27), the
regulation of which appears to ensure the simultaneous expression of negative (Ly49) and positive (NKR-P1) signaling molecules on NK cells and NK T cells.
c
mice as in
c+ mice, and in the absence of
NK-associated markers, the major determinant of NK T
cell selection remains the invariant V
14-J
281 TCR-
chain paired with the restricted TCR-
chains (10, 16). Although we cannot rule out a lower avidity reaction in the
absence of NKR-P1 or Ly49, we would suggest that the
expression of NK-associated markers are probably the result of additional maturation events after selection, rather
than being required for the selection process itself. This also
argues against the hypothesis that would make of the NK T
cells a peculiar lineage with a correlated expression of the
NK markers together with the V
14-J
281 invariant
chain. Indeed, the invariant
chain appears selected at the protein level rather than being produced through a genetic program that selectively recombines V
14 and J
281 (10).
c
NK Thymocytes.
thymocytes from
c+ or
c
mice were
cultured in vitro with soluble CD3 and antigen-presenting cells, IL-4 release could be detected in the supernatants
from
c+ and
c
cells (Table 1), although IL-4 production from
c
cells was relatively weak. We hypothesized
that one reason for the low IL-4 production from
c
thymocytes might relate to poor cell viability during the culture period (48 h). We have recently identified a novel cytokine, TSLP, which shares many functional similarities to
IL-7 (25), and uses the IL-7R
chain, but not the
c chain
for signaling, which can maintain
c+ and
c
thymocytes
in vitro (Park et al., unpublished data). As both
c+ and
c
NK thymocytes expressed the IL-7R
(Fig. 3), we added
TSLP to maintain thymocytes during the in vitro assay of
IL-4 production. Exogenous TSLP substantially increased
the amount of IL-4 produced from
c
NK thymocytes,
approximating the levels produced by
c+ cells under these
conditions (Table 1). Addition of TSLP to control thymocyte cultures from
2m
/
mice did not result in the
generation of IL-4. Therefore, TSLP can effectively substitute for IL-7 in stimulating NK thymocytes in vitro (28).
These results are in accord with the recent observations in
IL-7 knockout mice, in which NK thymocytes develop but
demonstrate a functional defect in IL-4 production after
CD3 stimulation (29). Exogenous IL-7 was able to restore
the IL-4 response in vitro (29). NK thymocytes from
c
mice also manifest abnormal IL-4 production in vitro in the
absence of IL-7R
engagement; however, this can be restored with TSLP (Table 1).
Experiment
Cells
IL-4
CD3 + APC
CD3 + APC + TSLP
U/ml
1
None
0
0
CD8
c+
70
120
CD8
c
20
110
2
CD8
2m
/
<5
20
CD8
c+
150
300
CD8
c
20
150
3
CD8
2m
/
ND
4
CD8
c+
ND
180
CD8
c
ND
180
NK thymocytes were isolated and stimulated as described in Materials
and Methods. Mice received 2 µg of anti-CD3 intravenously and splenocytes were prepared as described (13). IL-4 bioactivity was assayed
using the CT.4S indicator line.
The property of IL-4 production by NK T cells is likely
related to the selection by CD1 at a particular early ontogenic stage through the invariant V14-J
281 chain paired
with V
2, V
7, or V
8. This concept is supported by recent observations using V
14-J
281 transgenic mice, which
demonstrate an increased frequency of IL-4-producing NK
T cells resulting in increased basal levels of serum IgG1 and IgE (16). Our results are consistent with the idea that positive selection of the V
14-J
281-bearing TCRs confers
the IL-4-producing phenotype. Furthermore, we demonstrate that this unique ability of NK T cells to secrete IL-4
is not dependent on the expression (and therefore function)
of the NK-related molecules.
Next,
we examined whether c
NK thymocytes, despite their
phenotypic abnormalities, would be able to attain their
preferential localizations in the periphery. NK T cells normally comprise a small percentage of the lymphocytes
present in the spleen and lymph nodes (1); however, these
cells are abundant in the liver (20, 30). To quantitate peripheral NK T cells, lymphocytes from
c+,
c
, or
2m
/
mice were isolated from the liver and spleen and the amount
of V
14-J
281 mRNA was determined. NK T cells were
clearly present in the liver and spleens of
c+ mice (Fig. 4;
data not shown) as evidenced by the presence of V
14-
J
281+ mRNA. In contrast, levels of V
14-J
281+ mRNA
from
c
liver and spleen preparations were at or below
that of
2m
/
mice (which lack NK T cells) and well below that of
c+ controls (at least 200-fold less). Polyclonal
sequencing of V
14-J
281 amplicons from
c+,
c
, or
2m
/
samples showed an invariant sequence only in the
c+ samples (data not shown). Flow cytometric analyses confirmed the presence of NK1.1+ TCR-
int cells in intrahepatic lymphocytes from
c+ mice, which were not detected in preparations from
c
mice (Fig. 5). Lastly, in
vivo administration of anti-CD3 antibodies stimulated IL-4
release from cultured
c+ splenocytes, whereas no IL-4 production could be detected in splenocyte cultures from
c
mice (Table 2). Taken together, these results demonstrate
an absence of NK T cells in the liver and spleen of
c
mice.
|
Our results suggest that one or a combination of IL-2,
IL-4, IL-7, IL-9, or IL-15 is necessary for intrathymic maturation and the export/survival of the NK T cells to the
peripheral lymphoid organs. The coexpression of IL-7R
and IL-2R
chains on NK thymocytes suggest that IL-2,
IL-7, and/or IL-15 may be important in the final differentiation of these cells. Although IL-2-deficient mice have
reduced numbers of NK cells (31), NK thymocytes are present in IL-2
/
mice and have normal expression of the
NKR-P1 and IL-2R
. (Bendelac, A., personal communication). Moreover, IL-7-deficient mice display normal percentage of thymic and splenic NK T cells with a normal phenotype (29), and we have not detected a decrease in
V
14-J
281 transcripts in the thymus, spleen and liver of
IL-2
/
, IL-4
/
, or IL-7
/
mice compared with wildtype controls (Lantz, O., and J.P. DiSanto, unpublished
data). Taken together, these results fail to demonstrate the
essential role of either IL-2, IL-4, or IL-7 in the final maturation and export of NK thymocytes. However, functional
cytokine redundancy (the use of IL-15 in the absence of
IL-2, or TSLP in the absence of IL-7) may allow these processes to occur. Further studies using IL-2R
-deficient
mice (which can be considered as deficient in IL-2 and IL-15)
(32) and IL-7R
-deficient mice (which inactivate IL-7 and
TSLP) (33) should help to elucidate the
c-dependent interactions required for induction of NKR-P1 and Ly49 antigens on NK T cells and their export into the periphery.
Our results suggest a stepwise differentiation of NK T cell
which parallels that of mainstream TCR- development.
This is supported by the similarities between these two
lymphoid subsets: (a) both derive from a pool of early precursors requiring
c-dependent cytokines, because both are
reduced in absolute numbers by 20-fold in
c
mice; (b)
although NK T cells are selected on CD1 molecules and conventional T cells by classical MHC molecules, both
types of developing thymocytes exhibit TCR selection mechanisms that are independent of
c-cytokine interactions.
Thus, in contrast with classical NK cells, which fail to develop in
c
mice, NK T cells appear more closely related to
conventional TCR-
T cells. Complementary results from
Arase et al. (34) showed that NK1.1+ TCR-
T cells, as
well as mainstream
T cells, are absent in CD3-
-deficient mice, whereas NK cells were present.
Therefore, we favor a model of NK T cell development
in which recognition of CD1 at a certain stage of thymic
ontogeny (double-positive cortical thymocyte?) induces a
particular development program with the ability to secrete
IL-4 and the potential to express NK markers. The final acquisition of these NK markers (including members of the
NKR-P1 and Ly49 families) would require additional intrathymic maturation involving c-dependent cytokine interactions. We would hypothesize that induction of NK-associated markers on NK thymocytes might require signaling
through the IL-2R
(either IL-2 or IL-15), which despite
the expression of IL-2R
on
c
NK thymocytes would
not proceed in the absence of
c. It is not known whether
the absence of peripheral NK T cells in
c
mice is related
to (a) the absence of the NK markers, which would prevent their export to the periphery, (b) incomplete maturation not related to the NK phenotype, or (c) to their nonsurvival in the periphery due to their inability to respond to
c-dependent lymphokines. Furthermore, the precise molecular mechanisms that allow a TCR-mediated signal to
induce the acquisition of the NK markers or the ability to produce IL-4 only at a peculiar ontogenic stage remain to be
defined.
Address correspondence to Dr. James P. DiSanto, INSERM U429, Hôpital Necker, Pavillon Kirmisson, 149 rue de Sèvres, 75743 Paris, France.
Received for publication 6 November 1996 and in revised form 14 February 1997.
1Abbreviations used in this paper: DN, double negative;We thank L. Park (Immunex, Seattle, WA) for providing recombinant TSLP. We are indebted to D. GuyGrand and B. Rocha for discussions and for reviewing the manuscript, and to R. Murray (DNAX, Palo Alto, CA) and A. Bendelac for sharing unpublished results.
This work was supported by grants from the Association de la Recherche Contre la Cancer to O. Lantz and from the Ministère de la Recherche et de la Technologie (MRT) and the Institut National de la Santé de la Recherche Medicale (INSERM) to J.P DiSanto.
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