By
From the * Cancer Biology Program, Hematology/Oncology Division, Beth Israel Deaconess Medical
Center and Harvard Medical School, Boston, Massachusetts 02215; and Lymphocyte Biology
Section, Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital and
Harvard Medical School, Boston, Massachusetts 02115
A subset of human CD4CD8
T cells that expresses an invariant V
24-J
Q T cell receptor
(TCR)-
chain, paired predominantly with V
11, has been identified. A series of these V
24
V
11 clones were shown to have TCR-
CDR3 diversity and express the natural killer (NK)
locus-encoded C-type lectins NKR-P1A, CD94, and CD69. However, in contrast to NK
cells, they did not express killer inhibitory receptors, CD16, CD56, or CD57. All invariant
V
24+ clones recognized the MHC class I-like CD16 molecule and discriminated between
CD1d and other closely related human CD1 proteins, indicating that recognition was TCR-mediated. Recognition was not dependent upon an endosomal targeting motif in the cytoplasmic tail of CD1d. Upon activation by anti-CD3 or CD1d, the clones produced both Th1 and
Th2 cytokines. These results demonstrate that human invariant V
24+ CD4
CD8
T cells,
and presumably the homologous murine NK1+ T cell population, are CD1d reactive and
functionally distinct from NK cells. The conservation of this cell population and of the CD1d
ligand across species indicates an important immunological function.
The CD1 locus encodes a family of conserved nonpolymorphic proteins structurally related to MHC class I
and II proteins (1). Human and murine CD1-restricted
T cell lines and clones that recognize lipid antigens (6) or
hydrophobic peptide antigens (9) have been identified, indicating that CD1 proteins can function as specialized antigen-presenting molecules. A distinct function for murine
CD1d appears to be as a ligand or antigen-presenting molecule recognized by a population of T cells that express the NKR-P1C (NK1) cell surface C-type lectin (10). NK1
is otherwise restricted to NK cells and these NK1+ T cells
have also been referred to as NK T cells or natural T cells
(15, 16). Phenotypically, these cells are either CD4+CD8 Analyses of the CD1 genes in humans and other species
indicate that the proteins fall into two groups, CD1a-, b-,
and c-like (group 1), and CD1d-like (group 2) (3, 26). The
murine CD1 locus appears unique in that it has deleted the
group 1 genes, and contains only a duplicated group 2 gene
(27, 28). This observation suggests that there may be important functional differences between the CD1 proteins in
mice and other species. Nonetheless, a human invariant
TCR- To better understand the function of these cells and their
requirements for specific activation, a series of human invariant V Cell Lines and Clones.
T cell lines and clones were derived and
phenotypic analyses performed essentially as described (32). In
brief, DN V Antibodies and Phenotypic Analyses of T Cells.
The following antibodies were obtained from the fifth Leukocyte Workshop unless
otherwise indicated: anti-V
or CD4
CD8
(double negative, DN1), and forced expression of CD8 in transgenic mice results in the deletion
of this population (12, 17). Most strikingly, the majority of
these cells use an invariant TCR-
chain (V
14-J
281) that pairs preferentially with V
8, 7 or 2 (17). NK1+ T
cells appear to play a role in regulating immune responses, based upon their ability to rapidly produce large amounts
of IL-4 after stimulation with anti-CD3 in vivo (21).
However, these cells also produce large amounts of IFN-
,
and production of this cytokine can be specifically induced
by stimulation through NK1 (25). The immunological
functions of these cells and the physiologically relevant
CD1d-presenting cells mediating their activation remain to
be determined.
chain closely related to the murine invariant V
14-J
281 TCR has been identified as a predominant TCR
used by TCR-
/
DN T cells from multiple normal donors (29). This human invariant TCR-
is generated by a
rearrangement between V
24 (TCRAV24) and J
Q with
no N-region diversity. Subsequent studies have shown that
the human invariant V
24-J
Q TCR-
chain associates
preferentially with V
11 (TCRBV11) (30), which is
homologous to murine V
8. This invariant TCR may also
be expressed by a small proportion of CD4+ T cells, but
not CD8+ T cells (31). These observations suggest that human invariant V
24+ T cells are homologous to murine
NK1+ T cells.
24+V
11+ DN T cell clones were established
and characterized. TCR-
sequence analysis demonstrated
that these cells were derived from a polyclonal population
with no evidence of a shared
chain CDR3 motif. Phenotypically, the clones expressed high levels of NKR-P1A,
the only known human homologue of rodent NK1 (33).
They also expressed CD94 and CD69, two other C-type
lectins closely linked to NKR-P1A in a chromosomal region referred to as the NK locus. However, these cells did
not express the NK cell-associated p58 or p70 HLA class I
killer cell inhibitory receptors (KIRs; 34, 35) or other
markers of NK cells including CD16, CD56, or CD57.
Upon stimulation, the clones secreted cytokines associated
with both Th1 and Th2 cells, including IFN-
and IL-4,
respectively. Of the four characterized human CD1 proteins, each clone specifically recognized only CD1d expressed on human or hamster cell transfectants. CD1d recognition was not dependent upon the specific TCR-
chain CDR3 sequence. Moreover, deletion of an endosomal targeting sequence motif in the cytoplasmic tail of
CD1d (4, 5, 36) did not effect recognition, suggesting that
recognition by these cells was not dependent upon efficient targeting of CD1d to a specialized endosomal compartment
involved in antigen processing. These results demonstrate
that human invariant V
24+V
11+ DN T cells are a specialized population of CD1d-specific T cells. The results
also indicate that the similarities between this T cell population, and presumably the homologous murine NK1+ T cell
population, to NK cells may be limited to the expression of
certain closely linked NK locus-encoded C-type lectins.
24+V
11+ human peripheral blood T cell lines and
clones were established by sequential negative (CD4CD8) and
positive (V
24V
11) magnetic bead and FACS® sorting, respectively, of human peripheral blood T cells followed by stimulation
with PHA-P (reconstituted according to the supplier's instructions and used at a final dilution of 1:2,000; Difco, Detroit, MI)
and IL-2 (1.5 nM; Ajinomoto, Yokohama, Japan) in the presence of irradiated (5,000 rads) peripheral blood mononuclear cell feeders. Clones were established by limiting dilution. Clones and lines were maintained by restimulation every 3-6 wk as above with
phenotype monitored by FACS®. A human IL-2-dependent NK
cell line, NKL, (37) was provided by Drs. M. Robertson (Indiana
University Medical Center, Indianapolis, IN) and J. Ritz (Dana
Farber Cancer Institute, Boston, MA).
24 (C15B2) and anti-V
11 (C21D2)
(provided by Dr. A. Lanzavecchia [Basel Institute for Immunology, Basel, Switzerland]); anti-TCR-
/
(BMA031; provided by
Dr. R.G. Kurrle, Boehringwerke, Marburg, Germany); anti-CD3 (SPV-T3b and OKT3; provided by Dr. H. Spits [Netherlands Cancer Center, Amsterdam, Netherlands] and American Type Culture
Collection [Rockville, MD], respectively); anti-CD4 (OKT4;
American Type Culture Collection); anti-CD8
(OKT8; American Type Culture Collection); anti-CD8
(2ST8.5H7; from Dr.
E. Reinherz [Dana Farber Cancer Institute]); anti-CD16 (ABA2B1);
anti-CD28 (9.3); anti-CD56 (MEM188, MOC-1, 0218); anti-CD57 (TB01, TB02); anti-CD69 (PharMingen, San Diego, CA);
anti-CD94 (HP-3D9; PharMingen); anti-NKR-P1A (DX1 and
DX12, provided by Dr. L. Lanier (DNAX, Palo Alto, CA); HP-3G10 and 191B8); anti-p58 KIR (NK workshop mAbs GL183,
EB6, CH-L, and HP-3E4); and anti-p70 KIR (DX9; provided by
Dr. L. Lanier). Isotype control mAbs were P3 (IgG1), 4A7.6
(IgG2a, provided by Dr. D. Olive [Institut National de la Sante et
de la Recherche Medicale, Marseille, France]), and MPC11
(IgG2b).
CD1 Transfectants.
Chinese hamster ovary (CHO) and C1R
cells were transfected with a CD1d cDNA (3) in the pSR-neo
expression vector (39), followed by G418 selection and FACS®
to generate lines stably expressing CD1d. C1R cells stably transfected with CD1a, b, and c in the pSR
-neo vector were described previously (38). The CD1d-CD1a chimeric protein was
generated using an Xba1 restriction site located at the 3
end of
the
3 domain in CD1a and CD1d. The C1R line established
with this chimera construct in the pSR
-neo vector was uniformly CD1d positive after G418 selection and was used in these
experiments without further enrichment for CD1d+ cells.
Functional Analysis of T Cells. T cell activation for cytokine analysis was done using 105 T cells/well in 96-well flat-bottomed plates coated with anti-CD3 at 10 µg/ml for 48 h. Stimulation with CD1 was done using equal numbers of T cells and stimulator cells (105/well) for 48 h, unless otherwise indicated. For CHO cells, a 30-s glutaraldehyde fixation (0.05% in PBS) was used to bypass the need for costimulatory molecules (40). For stimulation by CHO and C1R transfectants, PMA was included at 1 ng/ml, except where stated otherwise. PHA control stimulations were carried out using a 2,000-fold dilution as described above. For antibody blocking experiments, CD1d transfectants were mixed with mAb dilutions immediately before addition of T cells.
Released cytokine levels were determined by ELISA with matched antibody pairs in relation to cytokine standards. The antibodies for IFN-Circulating DN T cells were isolated from the peripheral blood of two healthy donors (DN1 and DN2) by
anti-CD4 and -CD8 depletion (29, 32). DN V24+ T cells
were then positively selected from these populations using
the C15B12 mAb, specific for V
24 (41), and clones were established by limiting dilution. V
24+ T cell lines from
these populations were also established by bulk stimulation
with PHA, and the majority of the cells in these lines (75-
95%) were found to be V
11+ using the C21D2 mAb
(31). DN and single positive T cells from a third healthy
donor (DN3 and SP3, respectively) were isolated by positive selection with the V
24 and V
11 mAbs and cloned
by limiting dilution. As reported previously, the majority of
these latter clones were CD4+, but only the DN clones
from this donor expressed J
Q (32).
The TCR structure of a series of eight DN V24+
V
11+ clones from donor 2 and single clones from donors
1 and 3 was analyzed. Sequence analysis demonstrated that
the DN clones from donors 1 and 3 and all but one of the
clones from donor 2 expressed the invariant V
24 TCR-
chain (Table 1). Significantly, in one invariant V
24+
clone (DN2.C7), the V
24-encoded serine was apparently
removed during recombination and the serine was regenerated through an N-region addition and recombination 2 bp
further 5
in J
Q (Fig. 1). These results confirmed the high
frequency of the invariant V
24 TCR among DN V
24+
T cells, and suggested that this TCR is strongly selected
based upon its protein structure, rather than being generated exclusively by a developmentally programmed precise
joining of V
24 and J
Q gene segments.
|
In contrast to the invariant TCR- structure of the DN
V
24+V
11+ clones, multiple distinct TCR-
sequences
were identified (Table 1). The TCR-
sequences from a
series of CD4+, V
24+V
11+ clones that did not use the
invariant V
24 were also determined for comparison. The
identification of multiple V
sequences in the clones from
donor 2 demonstrated that the DN invariant V
24+ population may be derived from a large number of independent clones in single donors. The TCR-
chains were also
noteworthy for their markedly diverse CDR3 structures
and J
usage. There was no suggestion of a common CDR3
sequence motif, and even CDR3 length was quite variable.
This TCR-
diversity raised the possibility that these
clones might recognize diverse antigens in spite of their invariant TCR-
chain. Alternatively, the lack of conserved TCR-
chain structure may indicate that the TCR-
CDR3 did not contribute significantly to recognition by
the TCRs of these cells.
The relationship between invariant V24+ DN T
cells and NK cells was explored using a panel of mAbs recognizing proteins expressed predominantly by NK cells.
Humans appear to have only a single NK1-like gene, the
homologue of murine NKR-P1A, that is expressed by NK
cells and, at low levels, by a subpopulation of peripheral blood T cells (33). Nonetheless, NKR-P1A was expressed
at high levels by each of the invariant V
24+ clones (Table
2 and Fig. 2), but not by any of the CD4+ V
24+V
11+
clones that did not express the invariant V
24-J
Q rearrangement (data not shown).
CD69, CD94, and the NKG2 family are additional
C-type lectins linked to NKR-P1A in the human NK locus. CD69 is an early and transient T cell activation marker
(42), but expression of CD69 by each of the invariant
V24+ clones persisted at high levels for long periods (>3
wk) after in vitro stimulations (Table 2 and Fig. 2). CD94,
a possible NK cell receptor for class I proteins (43), was
also expressed at high levels by all of the invariant V
24+
clones. In contrast, prolonged high level expression of these NK locus-encoded proteins was not observed on any of a
series of similarly derived CD4+ clones or on more than a
very small fraction of PHA-stimulated PBLs (data not shown).
Cell surface expression of the NKG2 family (47), members
of which may associate with CD94 (48), could not be assessed as there are no NKG2-specific, surface-reactive NKG2
antibodies readily available (Bach, F., and J. Houchins, personal communications).
In contrast to the NK locus-encoded C-type lectins,
other functionally important receptors expressed by NK
cells, but encoded at other genetic loci, were not expressed
at significant levels by the invariant V24+ clones. These
included the p58 and p70 HLA class I-specific KIRs, CD16,
CD56, and CD57 (Table 2 and Fig. 2). Finally, each clone expressed low and variable levels of CD28 and CD8
, but
not CD8
. CD8
was similarly detected at low levels on
some cells from the CD4+ V
24+ invariant V
24
clones
(Table 2 and data not shown), consistent with its appearance as a late activation antigen. Taken together, these results further supported the conclusion that human invariant
V
24+ T cells and murine invariant V
14-J
281 NK1+
T cells are homologous. However, the relationship of these
cells to classical NK cells appeared limited to their expression of NK locus genes, but not of genes encoded elsewhere that are characteristically expressed by NK cells.
Cytokine production by the series of invariant V24V
11 clones in response to stimulation by plate-bound anti-CD3 was assessed. For comparison, a series of CD4+ V
24+-J
Q
V
11+
clones was also analyzed and IL-4/IFN-
ratios compared
(Table 3). With the exception of DN2.D7, the invariant
V
24+ clones all produced substantial levels of IFN-
and
IL-4, associated with Th1 and Th2 T cells, respectively.
Compared to the CD4+ clones, there was a trend towards
higher levels of IL-4 production by the invariant V
24+
clones, whether assessed based upon absolute IL-4 production or IL-4/IFN-
ratios. This contrasts, to some extent,
with an apparent bias in humans towards the generation of
T cells that produce much higher levels of IFN-
relative
to IL-4 (Th1 type cells) in the absence of polarizing stimuli
(49, 50). However, there was overlap between the invariant V
24+ DN clones and the CD4+ clones with respect
to IL-4 and IFN-
production, and IL-4/IFN-
ratio, indicating that the invariant V
24+ DN T cells had a phenotype that did not fall clearly into the Th1 or Th2 categories.
The DN clones also produced IL-13, and some produced
IL-10 at levels that were not clearly distinct from the
CD4+ cells.
|
CD1d recognition was assessed initially using CD1d-transfected CHO cells. Each of the five invariant V24+ clones assayed responded specifically to the
CD1d-transfected CHO cells based upon T cell proliferation (not shown) and cytokine release (Fig. 3). Recognition of CD1d required PMA and mild aldehyde fixation of
the target cells, which have been shown in other systems to
substitute for certain physiological costimulatory signals (40). Fig. 3 a shows that the CD1d transfectants stimulated IL-4 production from each of the clones except DN2.C9,
with the highest levels produced by DN2.B9 and DN2.D6.
The CD1d transfectants also strongly stimulated IFN-
production from three of these clones (DN2.B9, DN2.C9,
and DN2.D6) and modest, but specific, IFN-
release by
the other two clones (Fig. 3 b). In contrast, the invariant
V
24
clones did not respond specifically to the CD1d
transfectants (Fig. 3, a and b, and data not shown). The failure of the DN2.C9 clone to produce significant IL-4 in response to the CD1d transfectant was consistent with the
relatively low ratio of IL-4/IFN-
produced by this clone
in response to anti-CD3 stimulation (Table 3). Indeed, the
relative levels of IL-4 and IFN-
release in response to
CD1d from each of the clones were comparable to those
observed with anti-CD3 (Table 3).
Antibody inhibition studies were performed to confirm
that CD1d was recognized and to rule out the possibility
that peptide fragments of CD1d were the actual target of
the invariant V24+ clones. Two mAbs that recognized
native CD1d protein (42.1 and 51.1) could almost completely block activation of the DN2.D6 T cell clone at
concentrations
0.2 µg/ml (Fig. 4 and data not shown). A
third anti-CD1d mAb (68.2) partially blocked, but only at
higher concentrations. Several isotype-matched control antibodies used for blocking had no significant effect. Directly
comparable results were seen with the second clone analyzed (DN2.D5; data not shown).
Recognition of CD1d-expressing B Cell Transfectants.
Stimulation of invariant V24+ T cells by CD1d+ CHO cells required both PMA and aldehyde fixation, presumably due
to the absence of necessary costimulatory ligands on the
CHO cells. Although the target cells that mediate activation of invariant V
24+ T cells in vivo are not known,
normal B cells express CD1d (51) and may be a relevant
CD1d-presenting cell. Therefore, the C1R HLA-A and -B
negative B lymphoblastoid cell line (52), which does not
express detectable CD1d (our unpublished data), was used to confirm the results in CHO cells and to determine
whether the need for nonphysiological costimulation could
be reduced or eliminated. CD1d-transfected C1R cells
specifically stimulated each of the invariant V
24+ DN T
cell clones tested based upon IFN-
and IL-4 production and T cell proliferation (Fig. 5 a and data not shown), confirming the results in CHO cells. Stimulation did not require aldehyde fixation, but phorbol ester was still necessary.
The fine specificity of CD1d recognition was further assessed using C1R cells stably transfected with CD1a, b, or c
versus CD1d. The CD1a, b, and c transfectants were not
significantly more active than the mock transfectant in
stimulating IFN- (Fig. 5 b) or IL-4 production (not shown)
from any of the clones examined, although they were able to
stimulate CD1a-, b-, or c-reactive T cell clones, respectively (6, 53). In marked contrast, the CD1d C1R transfectants stimulated the production of IFN-
(Fig. 5 b) and IL-4
(not shown) at levels directly comparable to those produced in response to PHA.
Polyclonal DN T cell lines selected for expression
of V24 were sorted into V
11+ or V
11
populations and
examined to determine whether V
11 was necessary for
CD1d recognition. FACS® analyses showed that virtually all
of the cells in both lines were V
24+ (Fig. 6 a), and previous
RT-PCR analyses of these lines showed that both expressed
primarily or exclusively the invariant V
24 (32). The line
designated as DN2.V
11
had no significant V
11+ population, whereas the line designated as DN2.V
11+ was virtually all V
11+ (Fig. 6 a). It is of interest that cells in the
V
11
line consistently expressed slightly lower TCR levels, based upon staining with the anti-V
24 mAb (Fig. 6 a)
and anti-TCR mAbs (not shown). The relationship of this
observation to the preferential use of V
11 by invariant
V
24+ T cells is not clear, but could reflect greater stability
of the invariant V
24 when paired with V
11.
CD1 recognition by cells in both lines was compared using the panel of CD1-transfected C1R cells. Both the
V11+ and V
11
lines were activated specifically by the
CD1d-transfected C1R cells (Fig. 6 b). Although the response by the V
11
line was quantitatively less, the responses by both lines were comparable to the corresponding PHA responses and most likely represented maximal
activation for each line. These results demonstrated that
T cells expressing the invariant V
24 TCR-
paired with
V
s other than V
11 can mediate CD1d recognition.
Human CD1b, c, d, and murine CD1d, but not human CD1a, have short cytoplasmic tails containing a sequence motif, Tyr-X-X-Z (where X is any amino acid and Z is a hydrophobic amino acid), shown to regulate the intracellular trafficking of many transmembrane proteins (54). Previous studies demonstrated a critical role for this motif in the endosomal localization of human CD1b (36). To determine whether this sequence is necessary for the cell surface expression and function of CD1d, the transmembrane domain and cytoplasmic tail from CD1a was fused to the CD1d ectodomain. A C1R cell line transfected with this CD1d-CD1a chimera expressed moderate levels of CD1d at the cell surface (Fig. 7 a). The level of CD1d expression was lower than in the C1R line expressing wild-type CD1d, as this latter line was initially sorted for CD1d+ cells.
Despite lower levels of CD1d expression, the C1R cells
expressing the CD1d-CD1a chimeric protein were very effective at activating invariant V24+ clones. The DN2.D5
clone produced both IFN-
(Fig. 7 b) and IL-4 (not shown)
in response to the chimeric protein at levels comparable to
those induced by PHA stimulation. Identical results were
seen with a second clone, DN2.D6 (not shown). Therefore, deletion of the targeting motif did not impair CD1d
recognition by invariant V
24+ DN T cells.
This report demonstrated the expression of invariant
V24+ TCRs by a distinct population of CD1d-reactive
DN T cells that appear to be closely related, both phenotypically and functionally, to murine NK1+ T cells. A series of invariant V
24+V
11+ DN T cell clones were
shown to recognize CD1d expressed either by a hamster
(CHO) or human B cell (C1R) line. The fine specificity of
this recognition was demonstrated by the failure of these
clones to recognize CD1a, b, or c transfectants. Moreover,
antibody blocking studies indicated that intact CD1d,
rather than peptides derived from this protein, were recognized by these clones. These results provided strong evidence that CD1d recognition by these clones was mediated
directly by their TCRs.
The V11 chains from these clones were sequenced to
determine whether this population was monoclonal or
polyclonal and whether there were structural constraints on
the CDR3 region. This analysis revealed a unique V
11
chain with extensive N-region diversity in each of the
clones isolated from a single donor, demonstrating that
multiple independently derived clones contributed to this population. Sequence analysis of the V
24-J
Q chains also
revealed a clone with distinct codon usage in the V-J junction, indicative of N-region addition. These observations
suggest that the TCRs used by these clones are generated
through V-(D)-J recombination mechanisms and subsequent positive selection as with conventional T cells.
The V11 sequence analysis also revealed the lack of apparent structural constraints on the CDR3 region, since
there was marked variability in CDR3 length, J
usage,
and sequence. There was no suggestion of a sequence motif
that distinguished the V
11 CDR3 regions associated with
the invariant V
24 chain from those associated with other
noninvariant V
24 chains. This pattern of CDR3 independent recognition by one or several V
chains could be
consistent with selection or expansion by a superantigen
(possibly CD1d associated). However, conventional superantigen recognition is not dependent upon a V
chain
CDR3. It also appeared unlikely that these heterogeneous
V
11 chains mediated recognition of distinct CD1d-presented antigens since each clone recognized CD1d expressed by both hamster and human cells without the deliberate addition of an antigen. Therefore, the current data
suggest that V
11 may be structurally favored as an invariant V
24 partner and/or mediate direct contacts with
CD1d. However, pairing of the invariant V
24 with V
11
was not an absolute requirement for CD1d recognition since analysis of an invariant V
24+V
11
DN T cell line
demonstrated that the invariant V
24 can pair with other
V
s to generate CD1d-reactive TCRs.
Although CD1d recognition by multiple clones was demonstrated without the deliberate addition of an antigen, this did not rule out CD1d presentation of one or a small number of conserved ubiquitous endogenous or serum- derived antigens. CD1 has been shown to present lipid antigens (6, 53) and hydrophobic peptide antigens (9). Consistent with these observations, the crystal structure of murine CD1d reveals a potential deep hydrophobic antigen-binding cavity (55). The pathway(s) through which CD1 may acquire such hydrophobic or other antigens are not clear, but appear distinct from the MHC class I pathway (6, 56). Significantly, the cytoplasmic tails of human CD1b, c, and d, and murine CD1d contain a short tyrosine-based signal that has been implicated in trafficking from the plasma membrane to endosomal compartments (54), and a recent immunogold electron microscopy study demonstrated that a large fraction of CD1b molecules were located intracellularly in an endosomal compartment (36). Based upon these observations, one hypothesis has been that CD1 proteins are targeted to an acidic endosomal compartment and are there loaded with antigen.
To address this hypothesis in the case of CD1d, the cytoplasmic tail of CD1d was replaced with the cytoplasmic tail
of CD1a, which lacks recognizable targeting motifs. CD1a
proteins appear to traffic to the cell surface through the default secretory pathway and do not enter an endosomal
compartment (Sugita, M., M. Brenner, and S. Porcelli, unpublished data). The chimeric protein was recognized by
invariant V24+ DN T cell clones despite the loss of the
endosomal targeting signal, indicating that endosomal trafficking mediated by this signal was not necessary for CD1d
recognition by this cell population. Taken together, the
data in this report indicate that T cell recognition of CD1d
mediated by the invariant V
24 TCR may not involve a
specific antigen, although presentation of a conserved cellular antigen acquired through a pathway that is independent of the endosomal targeting motif cannot be excluded.
Moreover, it remains possible that CD1d presents specific
foreign antigens in vivo and that the in vitro responses reflect relatively low affinity interactions mediated by CD1d
binding to diverse nonspecific antigens.
In addition to specific antigen recognition by the TCR,
the activation of conventional T cells is dependent upon
the recruitment of p56lck to the TCR complex by the CD4
or CD8 accessory proteins. The invariant V24+ DN
clones express p56lck (data not shown) and it is very likely
that there are other accessory proteins that couple it to the
TCR in these cells. The consistent high level expression of
NKR-P1A by human invariant V
24+ DN T cell clones,
the expression of NK1 (NKR-P1C) by the homologous murine cell population, and the presence of a p56lck binding motif in the cytoplasmic tails of the murine NKR-P1
proteins make these clear candidate accessory proteins (59,
60). However, the presence of invariant V
+ T cells in
mouse strains that do not express NK1 or other NKR-P1 proteins argues against such a critical role for NKR-P1. It
should also be noted that the human NKR-P1A protein
does not contain the putative p56lck binding site (33) and,
in preliminary biochemical studies, we have been unable to
demonstrate an association between human NKR-P1A and
p56lck (Exley, M., unpublished data).
The human invariant V24+ T cell clones also expressed
two other C-type lectins, CD69 and CD94, encoded in a
chromosomal region that has been termed the NK locus.
CD94 may heterodimerize with NKG2 proteins (48), another family of C-type lectins encoded in the NK locus
(47), and this complex may be an MHC class I receptor
(44, 48). CD69 is an early and transient T cell activation antigen (42), but its expression by invariant V
24+ DN T
cell clones was persistent. This suggests that invariant V
24+ DN T cells may remain in an activated state longer
than conventional T cells. Alternatively, given the high
level of expression of other NK locus-encoded proteins
observed on invariant V
24+ T cells, CD69 expression
may reflect constitutive transcriptional activity of the NK
locus in these cells which is not directly related to T cell activation.
Although the expression of NKR-P1 and CD94 at high
levels suggests some relationship to NK cells, the invariant
V24+ DN T cells did not express a number of other molecules that play roles in NK cell function, such as CD16,
CD56, and CD57. In particular, the invariant V
24+ DN
T cells did not express p58 or p70 KIRs, although expression of family members not recognized by the multiple antibodies used here remains possible. Consistent with the
mAb data, preliminary RT-PCR amplification experiments with consensus KIR primers have similarly failed to
detect KIR expression (data not shown). This is in contrast
to recent data showing that KIRs may be expressed in
other T cell subpopulations (61). These observations
indicate that the link between invariant V
24+ DN T cells
and NK cells may be transcriptional activation of the NK
locus, with limited functional overlap between these cell populations.
Cytokine production by invariant V24+ DN T cells
was also analyzed and the results supported conclusions
reached in the mouse that these cells can produce significant levels of IL-4 in response to activation (20). However, they also produced other cytokines, particularly IFN-
,
at substantial levels, and their regulatory functions in vivo
are probably complex. Murine NK1+ T cells are responsible for the acute production of IL-4 in response to anti-CD3 in vivo (23), but this type of stimulus is clearly nonphysiological. It is unlikely that the function of this cell population is to determine systemic levels of IL-4 or other
cytokines, and more likely that the IL-4 produced in response to anti-CD3 stimulation reflects the primed activation state of these cells in vivo. A reasonable alternative hypothesis is that invariant V
24+ T cells function through
cell-cell interactions to provide individual CD1d+ target
cells with IL-4, IFN-
, or other cytokines that in turn direct the further proliferation and/or differentiation of these target cells.
B cells express CD1d (51) and represent one possible target cell for invariant V24+ T cells. However, CD1d may
also be widely expressed (51, 65) and invariant V
24+ T
cells may, therefore, have functionally important interactions with a number of different cell types. In particular,
the large fraction of T cells in murine bone marrow and
liver that are NK1+ (66, 67) presumably interact locally
with CD1d+ cells and may play roles in regulating B cell
maturation, myeloid development, or hepatic immune
function. Finally, recent reports indicate that loss of invariant V
24+ T cells in humans or of the homologous NK1+
T cell population in mice is associated with disease progression in several autoimmune diseases (68). Although the
precise functions of invariant V
24+ T cells and their murine homologues remain to be clarified, the conservation of
this cell population and of the CD1d ligand across species
suggests an important immunological function.
Address correspondence to Steven Porcelli, Lymphocyte Biology Section, Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital and Harvard Medical School, 250 Longwood Ave., Boston, MA 02115; Phone: 617-432-4984; FAX: 617-667-0610; or Steven P. Balk, Hematology/Oncology Division, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215. Phone: 617-667-0600; FAX: 617-667-0610.
Received for publication 7 March 1997 and in revised form 21 April 1997.
For antibodies and cell reagents we wish to thank Drs. L. Lanier, A. Lanzavecchia, M. Robertson, J. Ritz, H. Spits, E. Reinherz, D. Olive, and R.G. Kurrle. We also thank other members of the Lymphocyte Biology Section and Geoffrey Sunshine for advice, and Alexis Fertig for technical assistance.This work was supported by National Institutes of Health grant R01-AI33911 to S.P. Balk and National Institutes of Health grant R01 AI40135 to S. Porcelli. S. Porcelli was also supported by an Investigator Award from the Arthritis Foundation.
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