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*
Department of Microbiology and Immunology and the Molecular Biology Institute, University of California at Los Angeles, Los Angeles, CA 90095;
Department of Medicine and Microbiology Immunology and Howard Hughes Medical Institute, University of California at San Francisco, San Francisco, CA 93143; and
Department of Immunology, Department of Cellular and Molecular Biology, Lund University, Lund, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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14, the major TCR expressed by NK1+ T cells,
exhibited greatly diminished reactivity to mutant CD1 molecules that do
not traffic through endosomes, although the reactivity of other T cell
hybrids to this mutant was not greatly affected. Therefore, we propose
that at least some of the autoreactive T cells require endosomally
derived CD1-bound ligands, and that they are capable of distinguishing
between a diverse set of such self-ligands, which might be either
autologous lipoglycans or peptides. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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On account of their association with ß2-microglobulin, CD1 molecules are considered to be class I like. By contrast to class I molecules, however, the surface expression of CD1 is transporter associated with Ag processing (TAP) independent (3, 4, 5, 6). Most CD1 molecules have a tyrosine-containing sequence in their intracytoplasmic domain that could serve as an endosomal localization sequence. This type of targeting signal has been shown to be responsible for the rapid internalization of endocytic receptors from the plasma membrane, as well as for protein targeting to various endosomal compartments following exit from the trans-Golgi network (7, 8). Consistent with a possible endosomal localization of CD1 molecules, mycolic acid and lipoarabinomannan presentation by human CD1b molecules could be inhibited with agents that interfere with endosomal trafficking (9, 10). In addition, human CD1b could be localized in a variety of endosomal structures, including late endosomes, lysosomes, and the MHC class II-positive compartments where it colocalizes with lipoglycan Ags (11, 12). Removal of the putative endosomal localization motif from CD1b led to a redistribution of the molecules with reduced expression in vesicles and a predominant localization to the cell surface (11). Collectively, these data indicate that CD1 molecules, particularly CD1b, are distinct in their intracellular trafficking from other class I molecules, and they strongly suggest that endosomes are involved in the presentation of Ags by CD1. mCD1 has not been shown to be located in endosomes, and a requirement for the endosomal localization motif for Ag presentation has not been demonstrated to date for any CD1 molecule.
The question remains open as to what types of Ags mCD1 may present. The ability of mCD1 to bind peptides with a hydrophobic sequence motif, and to present these peptides to T cells, has been clearly demonstrated (13). Despite this, the groove of mCD1 is substantially narrower than that of class I and class II molecules (14) and it is not certain how long peptides, which are required for optimal mCD1 binding (13), could fit into it. Human CD1b and CD1c molecules have been shown to present lipid-containing Ags from mycobacteria (9, 10), suggesting a possible alternative type of ligand for mCD1. Interestingly, some electron density was found in the groove of soluble mCD1 molecules derived from insect cells, although the nature of this material remains to be determined (14). In summary, it is possible that mCD1 can bind both peptide and nonpeptide Ags, but the structural basis for these interactions are still unknown.
While there is convincing evidence for Ag presentation by CD1
molecules, it is surprising that many of the CD1-reactive T cells
described in both mice and humans are reactive for autologous CD1
molecules (15, 16, 17, 18, 19, 20). In mice, this population includes the majority of
so-called natural T cells (21), which express markers typical of NK
cells. Many but not all of these mouse NK T cells express a TCR
containing an invariant -chain that is V14-J281,
preferentially paired with one of three ß-chains (22). A second
source of mCD1-autoreactive T cells is found in the residual population
of CD4+ T cells in MHC class II-deficient mice (16). This
set of mCD1-reactive lymphocytes expresses a diverse set of TCRs (16).
As these cells have been studied mostly as T cell hybridomas, it is not
known if they are truly autoreactive in vivo, and if so, why these
mCD1-autoreactive T cells are not subject to self-tolerance
induction. Their function also is unknown, although it has been
proposed that early expression of IL-4 by NK1+ T cells
could stimulate Th2-type immune responses (23, 24, 25). Other functions for
NK T cells in the regulation of autoimmune disease and the surveillance
for tumors also have been proposed (26, 27, 28, 29, 30).
In this report, we have assessed the heterogeneity of mCD1-autoreactive T cells. A panel of five T cell hybridomas was tested with a series of different cell types that express defined quantities of the identical mCD1 molecules. We find that each of the hybridomas exhibits a different reactivity pattern, and that their reactivity is cell type dependent, rather than being dependent upon the amount of mCD1 expressed. In addition, we have determined that mCD1 is found in endosomes, and have defined its endosomal localization sequence. Mutant mCD1 molecules unable to enter the endosomal pathway are poor stimulators of one of the hybridomas, suggesting that at least some of the mCD1-autoreactive T cells require an mCD1 binding ligand found in the endosomes of autologous cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The derivation and characterization of the mCD1-autoreactive
hybridomas 19, 68, and 24 have been described previously (16). These
hybridomas originated from the remnant CD4+ splenic T
cell population in class II-deficient mice. Hybridomas DN3A4-1-4 and
DN3A4-1-2 are derived from C57BL/6 NK1+ thymocytes. Their
derivation will be described in detail elsewhere (Brossay et al.,
manuscript in preparation). Briefly, sorted NK1+ thymocytes
were stimulated in vitro with plate-bound anti-TCR ß clone
H57-597 mAb, grown in complete RPMI 1640 medium with 104
U/ml of rIL-2 (PharMingen, San Diego, CA) and 6 x 103
ng/ml of IL-7 (PharMingen) and fused with TCR ß-
BW5147 cells according to standard protocols (31). TCR ß-chain
expression was determined by flow cytometric analysis using specific
mAbs, and V14-J281 mRNA expression was determined by reverse
transcriptase-PCR. For the stimulation assays, 5 x
104 T hybridoma cells per well were cultured in the
presence of 3 x 105 CD1+ or control
stimulator cells. After 20 h, IL-2 release was evaluated in a
sandwich ELISA using rat anti-mouse IL-2 mAbs (PharMingen).
Mice and cell lines
C57BL/6 strain mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice that were 8 to 12 wk old of both sexes were used. All mice were housed under specific pathogen-free conditions in the UCLA Center for Health Sciences Vivarium. B cell lymphoma A20 cells were obtained from the American Type Culture Collection (Rockville, MD). RMA-S cells, J774 cells, and mCD1-derived transfectants have been described elsewhere (1, 4).
mCD1 site-directed mutagenesis
For all the PCRs, pHßAprneo-CD1.1 (4), which contains a
full-length mCD1.1 cDNA cloned behind the ß-actin promoter, was used
as a template. PCR was performed using 1x PCR buffer, 2.5 mM
MgCl2, 200 µM dNTP, 1 µM primer, and 1.5 U
Taq polymerase (Perkin-Elmer Cetus, Branchburg, NJ). Samples
were heated at 94°C for 3 min followed by amplification for 15 cycles
of 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C. The 5' primer
for both constructs was 5'CAC GTC GAC ACA TGC GGT ACC TAC CAT GGC TG.
The tail-deleted construct (TD) was obtained by introducing a stop
codon at position 6 of the cytoplasmic domain (Fig. 2
A) using 3' primer 5' GAT GGA TCC TGT CAA GCG CTT
CTC CTT C. The Tyr
Ala construct (YA) was made by
oligonucleotide-directed mutagenesis using PCR and 3'primer 5'GA TGG
ATC CTG TCA CCG GAT GTC TTG AGC AGC GC (mutant alanine codon
in bold).
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Final PCR products were purified by agarose gel electrophoresis and ligated into the TA cloning vector (Invitrogen, Carlsbad, CA). Clones were then sequenced using the dye primer cycle sequencing kit with the ampli-Taq DNA polymerase FS (Perkin-Elmer Cetus). For expression in mammalian cells, mCD1.1 cDNA was inserted into the unique BamHI and SalI sites of the mammalian expression vector pHßAprneo, which contains the human ß-actin promoter. Twenty micrograms of plasmid was linearized and electroporated (Gene Transfector 300; BTX, San Diego, CA) into 107 A20 B lymphoma cells. Geneticin (G418; Life Technologies, Gaithersburg, MD) selection was begun at 48 h postelectroporation. Stable transfectants were chosen at 3 to 4 wk and stained with 1B1 mAb to detect mCD1 surface expression.
Flow cytometric analysis
The anti-mCD1 mAb 1B1 has been previously described (1, 5) and was biotinylated using a kit from Pierce Chemical (Rockford, IL). Secondary reagents for mCD1 detection were streptavidin coupled to phycoerythrin (PE) or tricolor (Caltag, South San Francisco, CA). For staining, cells were suspended in buffer composed of PBS, pH 7.3, containing 2% BSA (w/v) and 0.02% NaN3 (w/v). Cells were first incubated with 2.4G2 mAb (PharMingen) for blocking Fc receptors, incubated at 4°C for 20 to 30 min with the primary Ab, washed twice, and then further incubated with secondary reagents for another 20 to 30 min at 4°C. After two washes, the cells were resuspended in Fix buffer consisting of PBS, pH 7.3, 0.02% (w/v) NaN3, and 1% (w/v) paraformaldehyde (Sigma, St. Louis, MO). Cells were analyzed on a Becton Dickinson (Mountain View, CA) FACScan 440 flow cytometer (located at Jonsson Cancer Center Flow Cytometry Core Facility, UCLA).
Confocal microscopy
A20 cells and transfectants were plated in eight-well permanox chamber slides at a density of 1 x 104 cells per well. After overnight adherence, mCD1 molecules were immunofluorescently localized in formaldehyde-fixed cells that were permeabilized with 0.2% saponin (Sigma) in PBS for 25 min. Before the addition of the primary Abs, the cells were incubated with 10% donkey serum in PBS containing 0.05% saponin followed by anti-Fc mAb 2.4G2. mCD1 molecules were detected with biotinylated 1B1 mAb followed by streptavidin coupled to FITC (Jackson ImmunoResearch, West Grove, PA). For the colocalization experiment, cells were stained with both rabbit anti-H2-M polyclonal Abs (kindly provided by Dr. Ira Mellman, Yale University, New Haven, CT) and biotinylated 1B1 mAb, followed by FITC-labeled donkey anti-rabbit IgG and streptavidin coupled to Texas Red (Jackson ImmunoResearch). The fluorescently labeled cells were analyzed with a Leica CLSM confocal laser scanning microscope (Deerfield, IL) using the x63 objective.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To test for heterogeneity among mCD1-autoreactive T cells, we used
a panel of T cell hybridomas. The members of this panel can be
classified into two groups (Table I). The
first group, including T cell hybridomas 24, 19, and 68, consists of
lymphocytes derived from the sorted CD4+ population
present in the spleen of class II-deficient mice. These hybridomas do
not express V14 mRNA (16). The two members of the second group,
DN3A4-1-2 and DN3A4-1-4, were obtained from the enriched
NK1+ population from the thymus of a normal C57BL/6 mouse,
and they express V14 mRNA along with either Vß8.2, which is
typical of this NK1+ population (22), or Vß10 (our
unpublished observations).
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, hybridoma DN3A4-1-2 reacted more
strongly to autologous C57BL/6 thymocytes than it did to autologous
splenocytes, despite approximately equivalent average levels of mCD1
expression when cells from these two organs are compared (mean
fluorescence intensity 178 for spleen and 146 for thymus). Each
stimulator population was tested for the level of mCD1 expression on
the day of the assay. Despite this nearly equivalent average expression
level, with spleen being slightly greater, the level of IL-2 release
was more than sixfold higher when thymocytes were used as stimulators
for this T cell hybridoma, a result that was typical of four different
experiments (average of 5.4 times more IL-2). The tissue preference of
DN3A4-1-2 is consistent with its derivation from the thymus, and with
the possibility that T cells with this receptor are stimulated by Ag in
vivo. By contrast, the other thymus-derived hybridoma, DN3A4-1-4, does
not distinguish between mCD1+ cells from these two tissues.
Conversely, hybridoma 24, which is derived from splenocytes, reacted
more strongly to spleen cell-derived stimulators than to thymocytes
(Table I). The threefold increase in IL-2 release obtained with splenic
stimulators was typical of the results from three different experiments
(average of 2.5 times more IL-2), suggesting that this hybridoma may be
more reactive with B cells than with T cells. Consistent with this,
hybridoma 24 reacted to purified splenic B cells, but only weakly or
not at all to splenic T cells (data not shown). Unlike hybridoma 24,
splenocyte-derived hybridomas 68 and 19 did not react more strongly to
splenocytes, although hybridoma 19 reacted more strongly to both cell
types than did hybridoma 68. Because some T cell hybridomas react more
strongly to thymocyte stimulators, while others react more strongly to
splenic stimulators, these data demonstrate that the cells from one
mCD1+ population are not intrinsically better at activating
the mCD1-autoreactive T cell hybridomas. Different patterns of reactivity of mCD1-autoreactive T cell hybridomas to mCD1-transfected cells
The heterogeneity of mCD1-autoreactive T cell hybridomas was
further examined using transfected cell lines. The analysis of mCD1
expression by these transfectants is presented in Figure 1. Each cell line was transfected with a
ß-actin promoter-driven CD1.1 expression construct, drug-resistant
clones were obtained, and high expressors for wild-type mCD1 then were
selected for further analysis. The transfected cell types include a
thymoma (RMA-S), a B cell lymphoma (A20), and a macrophage-like
tumor (J774).
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, several of the T
cell hybridomas responded differently to this set of three
transfectants. CD1.1-transfected J774 cells induced an IL-2 release
equivalent to transfected RMA-S cells by hybridoma 24, despite a more
than fourfold lower expression of mCD1 when the linear scale
fluorescence intensities of the transfectants are compared.
Furthermore, hybridoma 24 produced more than 10 times more IL-2 in
response to CD1.1-transfected A20 cells when compared with
CD1.1-transfected RMA-S cells (Table II, representative data from five
experiments). Although the transformed cell types may not be completely
representative of their nontransformed counterparts, the data obtained
with A20 and RMA-S transfectants correlates with the stronger
reactivity of the 24 hybridoma to splenocytes than to either thymocytes
or splenic T cells. By contrast to hybridoma 24, hybridomas 68 and 19,
also splenocyte derived, responded similarly to CD1 transfectants of
A20, J774, and RMA-S cells, with only a slightly greater response to
A20 transfectants.
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14-J281 TCR -chain
typical of NK1+ T cells, exhibits a slightly different
reactivity pattern to the transfected cells. Although it responded to
all three CD1.1-transfected cells, the response to RMA-S transfectants
was nearly 10-fold lower than the response to A20 transfectants.
Finally, the responses of DN3A4-1-4 were lower than those of the other
hybridomas. While this could reflect chromosome loss or other factors,
a comparison of the relative response to different cell types revealed
yet another pattern of reactivity. The J774 macrophage transfectant was
relatively ineffective at stimulating this hybridoma, although
mCD1+ J774 cells could stimulate the remaining four
hybridomas quite well.
The combined results for the five mCD1+ stimulator cell
types, including splenocytes, thymocytes, and the three transfected
cells are presented in Table III. The
table shows only the rank order of the different stimulators for each
hybridoma. Several conclusions can be drawn from this summary table and
the quantitative data in Tables I and II. First, each of the five
hybridomas has its own reactivity pattern, although the differences
between some of the pairs of T cells are subtle. For example,
hybridomas 19 and 68 respond very similarly to the three transfected
cell types, but 19 reacts much more strongly to spleen cells and
thymocytes. Second, as noted above, the response does not correlate
well with the level of mCD1 expression. A T cell responsive only to the
level of mCD1 expression would react best to A20 transfectants,
followed by RMA-S transfectants, with the response to splenocytes,
thymocytes, and J774 transfectants being approximately equivalent. None
of the T cell hybridomas displayed this pattern. The relatively poor
ability of CD1-transfected RMA-S cells to stimulate the T cell
hybridomas is not due to their TAP deficiency, because thymocytes from
TAP-deficient mice stimulate the T cell hybridomas as well as
thymocytes from wild-type mice (data not shown). Third, while the level
of mCD1 clearly is not the prime determinant of T cell hybridoma
responsiveness, it is likely to be a significant factor. All of the
hybridomas respond well to the A20 transfectant, which has the highest
levels of expression. Furthermore, the relatively weak response of
hybridoma 24 to splenic T cells and thymocytes can be partially
overcome in transfected RMA-S T cells that express high levels of mCD1.
The level of mCD1 expression by untransfected RMA-S cells (Fig. 1) is
not sufficient to stimulate the 24 T cell hybridoma.
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The heterogeneity of reactivity patterns exhibited by the mCD1-autoreactive T cell hybridomas is consistent with the possibility that each clone of autoreactive T cells recognizes one of a diverse set of mCD1-bound ligands. To investigate whether this might be the case, we attempted to disrupt the intracellular trafficking pattern of wild-type mCD1 by site-directed mutagenesis to determine whether the ability of autoreactive T cell hybridomas to recognize mCD1 is affected by such mutations. Human CD1b molecules are found in a variety of endosomal structures, predominantly various types of late endosomes (11, 12), and the endosomal localization of these molecules is dependent upon the YQDI sequence motif in the intracytoplasmic tail (11). mCD1 contains a similar sequence motif in its cytoplasmic tail, suggesting that it also may be found in endosomes.
We analyzed wild-type mCD1 transfectants of A20 cells for the
intracellular localization of mCD1. A20 B lymphoma cells were chosen
for transfection because they express relatively high levels of class
II-containing vesicles (CIIV), compartments specialized for class II
peptide loading (32). Furthermore, they could be representative of a
physiologically relevant mCD1 APC. Although nearly all normal mouse B
cells are positive for mCD1 (1), before transfection, A20 cells are
negative upon staining with the 1B1 mAb (Figs. 1 and 2B). As
shown in Figure 3, A and D, wild-type mCD1 is
located on both the cell surface and intracellular vesicles in
transfectants. mCD1 colocalized extensively with H-2 M (mouse DM)
molecules intracellular vesicles (Fig. 3, D-F), suggesting that mCD1 also may be
found in the CIIV and in lysosomes, as described for human CD1b. We
currently are carrying out a detailed study of mCD1 expression in
different endosomal compartments (our unpublished data).
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To disrupt the endosomal localization of mCD1, we constructed two
mutants in the mCD1 cytoplasmic tail (Fig. 2A). One has the
COOH-terminal five-amino acid sequence from the intracytoplasmic tail
of mCD1 deleted (mCD1TD), and the second has the tyrosine substituted
by an alanine (mCD1YA). As can be seen in Figure 2B,
neither deletion of the endosomal localization signal, nor substitution
of the tyrosine with an alanine affected the ability of mCD1 to be
expressed on the surface of A20 cells. The figure shows the flow
cytometry profiles of transfected A20 cells that expressed surface
levels of mutant mCD1 molecules higher than (mCD1TD), or equivalent to
(mCD1YA), the levels obtained in the wild-type mCD1 transfectants.
Confocal microscopy was used to examine the intracellular distribution
of the tail-deleted, and YA-substituted forms of mCD1 in optical
sections of transfected A20 cells. Permeabilized A20 cells expressing
either the mCD1TD or the mCD1 YA mutants revealed homogenous
staining around the rim of the cell that is characteristic of cell
surface staining. There was only a low level of intracytoplasmic
staining, indicating a redistribution of mCD1 from endosomal vesicles
to the cell surface in the mutants (Fig. 3, B and C).
These data are consistent with those found for the human CD1b protein,
which does not traffic to endosomes when the cytoplasmic
tyrosine-containing sequence motif is deleted (11), and they also
demonstrate that the tyrosine is critical for this process. In
addition, we also show that the tail-deleted form of mCD1 does not
colocalize with H-2 M molecules (Fig. 3, G–I).
mCD1 molecules that do not localize to endosomes can stimulate some mCD1-autoreactive hybridomas
We analyzed the ability of the mCD1 cytoplasmic tail mutants to
stimulate mCD1-restricted T cells. The mCD1 specificity of these cells
was confirmed using anti-CD1 mAb 1B1, which completely blocks the
stimulation of the five T cell hybridomas used in this study (Table IV). Table IV shows that truncation or
mutation of the mCD1 endosomal localization signal is not critical for
the activation of T cell hybridomas 19, 24, 68, and DN3A4-1-4. Indeed,
they produce similar levels of IL-2 upon stimulation with either the
mCD1-, mCD1TD-, or mCD1YA-transfected cells. By contrast, the
reactivity of hybridoma DN3A4-1-2 was clearly affected by these
mutations. The tail-deleted form stimulated 3.5-fold less IL-2 release,
while mCD1YA stimulated sevenfold less IL-2 release. These data are
representative of seven experiments with an average decrease of 3.3 for
mCD1TD and five experiments with an average decrease of 6.1 for
mCD1YA.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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helices, or whether they are
dependent upon CD1-bound ligands.
In this study, we have found a striking degree of heterogeneity in the
ability of autoreactive T cell hybridomas to respond to different
mCD1+ T cells. When tested against a panel of five
different stimulators, each of the hybridomas exhibited a different
reactivity pattern, with the differences in the level of IL-2
production obtained varying by more than an order of magnitude. The
results from two previous studies suggested that there was some degree
of heterogeneity in the ability of mCD1-autoreactive T cell hybrids to
respond to mCD1+ stimulator cells (15, 16). Bendelac et al.
reported that one T cell hybridoma from NK1+ T cells that
expresses a Vß8/V14 TCR reacts with thymocytes but not with
splenocytes (15). The thymocyte preference of these cells is in
agreement with the results presented here from an additional
NK1+-derived T cell hybridoma. Furthermore, we previously
reported that the mCD1-autoreactive T cell hybridomas from a class
II-deficient mouse exhibit significant heterogeneity with regard to
their ability to react with splenocytes from different mouse strains
(16). Neither of the previous studies, however, reported on the level
of mCD1 expression by the different stimulator cell types, and for the
most part, complex populations of APCs were used. Furthermore, in our
previous work, some of the heterogeneity in the mCD1-autoreactive T
cells could have been due to mCD1 polymorphisms in different wild mouse
strains (16). In this study, we used both transfected cell lines as
well as cell populations to demonstrate a significant degree of
heterogeneity of mCD1-autoreactive T cells, and we were able to
demonstrate that this heterogeneity is not related primarily to the
level of mCD1 expression.
In light of these findings, several explanations for the diversity in recognition by the mCD1-autoreactive T cells can be considered. First, the differential ability to recognize the product of the CD1.2 gene (the second CD1 gene in mice, which is closely related to CD1.1 (34)), could be an important factor. This is unlikely, however, because C57BL/6 thymocytes and splenocytes were used in this study, and this strain has been reported to have a defective CD1.2 gene (35). Furthermore, the mCD1 mAb 1B1 is incapable of recognizing a transfectant known to be expressing CD1.2 on the cell surface (our unpublished observations), and therefore is considered to be CD1.1 specific. This mAb could, however, completely block the stimulation of the five T cell hybridomas used in this study, demonstrating the CD1.1 specificity of this reactivity. Second, differential requirements for costimulatory molecules could be involved. This could include either B7-1 and B7-2, or other molecules, some of which may not have been completely characterized (36). We consider this unlikely because of the variety of reactivity patterns obtained from a small number of cells, and because T cell hybridomas are usually costimulatory independent. Third, it is possible that posttranslational modification of mCD1 molecules, particularly glycosylation, could play a role in the stimulation of mCD1-autoreactive hybridomas. There are no data that argue strongly against this possibility. The fourth alternative explanation, which we consider the most likely one, is that each of the hybridomas recognizes a different member of a set of diverse, and perhaps cell type-specific, mCD1-bound, autologous ligands. Consistent with the requirement for particular mCD1-bound ligands, we were not able to stimulate a significant amount of IL-2 release when the hybridomas were cultured with microwell plates coated with soluble, purified mCD1 molecules purified from Drosophila melanogaster tissue culture cells (13) or with D. melanogaster transfectants (4) that express mCD1 on the cell surface (data not shown). Although the groove of soluble mCD1 obtained from insect cells contains some electron density, which is likely to be an unidentified ligand (14), this material presumably is not capable of stimulating mCD1-autoreactive T cells.
mCD1 has been shown to present hydrophobic peptides (13), but
there are to date no Ags to be presented by mCD1 that are known to
require internalization and processing. Nevertheless, by analogy with
CD1b (9, 10, 12, 37), and given the presence of a YQDI endosomal
localization motif sequence in the cytoplasmic tails of both CD1b and
mCD1, we considered it likely that the loading of Ags onto the mCD1
groove occurs in endosomal vesicles. In this report, we have
demonstrated that mCD1 is in fact localized to endosomes, with
extensive colocalization with late endosomal markers (Tangri et al.,
manuscript in preparation) and mouse H-2 M, which marks the CIIV. We
also have demonstrated that targeting of mCD1 to endosomal compartments
is controlled by the YQDI cytoplasmic tail motif, and that the tyrosine
is critical for this endosomal localization, as it is in several other
proteins besides CD1 molecules (8, 38). Consequently, one might predict
that deletion or mutation of the endosomal localization sequence should
either decrease or shut down the mCD1 Ag-loading process. We were
therefore surprised to find that disruption of the mCD1 endosomal
localization signal affected the reactivity of only one of five
mCD1-autoreactive T cell hybridomas, and that even in this one case,
there was not a complete loss of reactivity. Assuming that a diverse
set of self-ligands are recognized by the autoreactive T cell
hybridomas, these data suggest that autologous ligands can be loaded
into mCD1 molecules outside of endosomes, perhaps in the endoplasmic
reticulum or on the cell surface. Alternatively, we cannot rule out the
possibility of a rapid transit of the mutated mCD1 molecules through
endosomes, but this also probably would not allow an efficient Ag
loading. Finally, as noted above, some of the hybridomas may be
reactive mostly with the mCD1 helices, and therefore they are
perhaps sensitive to any carbohydrate modification of the protein back
bone, in which case they might not be highly ligand dependent.
Interestingly, the one hybridoma partially dependent upon the endosomal
localization signal, DN3A4-1-2, expresses a Vß8/V14 TCR. This is
the TCR characteristic of the NK1+ T cell population, which
has been proposed to play an important role in immune regulation. A
similar T cell subset has been found in humans, which predominantly
expresses a V24-JQ and Vß11 TCR (33, 39, 40), the human
homologues of V14 and Vß8. Recently, it has been shown that some
of these Vß11/V24 T cells react with human CD1d, the closest human
homologue of mCD1 (20). In humans, Exley et al. have shown that a
chimeric protein, in which the CD1d cytoplasmic domain has been
replaced with the cytoplasmic tail of CD1a, was recognized by invariant
V24+ double negative T cells (20). Because the
cytoplasmic tail of CD1a lacks recognizable targeting motifs, these
authors also considered the possibility that the recognition of human
CD1d by autoreactive T cells may not involve a specific CD1d-bound Ag.
The results from human T cells appear to be inconsistent with ours,
indicating that a mouse T cell expressing Vß8 and V14 exhibits a
partial requirement for the mCD1 cytoplasmic endosomal localization
sequence. It is possible, however, that there is heterogeneity for
CD1-bound recognition even within the lymphocyte subset with
"invariant" TCRs, as different ß-chains and ß-chain junctional
regions could mediate the recognition of different ligands. Only one
clone (V14/Vß8) was tested, and similarly only two human T cells
(V24/Vß11) were assayed for recognition of the human CD1d chimeric
protein. Tests of larger panel of cells might reveal heterogeneity. A
role for the ß-chain in determining the fine specificity of mCD1
autoreactivity is indicated by comparing the results obtained with
hybridomas DN3A4-1-2 and DN3A4-1-4, which express identical -chains
with different ß-chains. Most notably, unlike the Vß8+
hybridoma DN3A4-1-2, Vß10+ hybridoma DN3A4-1-4 does not
require the mCD1 endosomal localization signal, and it cannot respond
well to J774 transfectants. An alternative and less likely explanation
for the differing results in mouse and human is that there is a
systematic difference between the Ags recognized by mouse and human
autoreactive T cells, despite the use of homologous TCRs to recognize
homologous Ag-presenting molecules.
In summary, our data demonstrate that autoreactive T cells distinguish between different types of mCD1-expressing cells, and they suggest that a diverse set of different self-ligands might be responsible for this variability. We have shown that mCD1 relies upon a tyrosine-containing motif in its cytoplasmic tail to localize to endosomes including the CIIV. Furthermore, our data demonstrate that endosomal localization is important for the stimulation of a minority of mCD1-autoreactive T cells, suggesting that an endosomally derived ligand might be important for the stimulation of some of these T cells.
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Acknowledgments |
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Footnotes |
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2 Current address: La Jolla Institute of Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121.
3 Address correspondence and reprint requests to Dr. Mitchell Kronenberg, La Jolla Institute of Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121. E-mail address:
4 Abbreviations used in this paper: mCD1, mouse CD1; CIIV, class II-containing vesicles; TD, tail-deleted construct; PE, phycoerythrin; MFI, mean fluorescence intensity.
Received for publication October 21, 1997. Accepted for publication December 11, 1997.
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ß+ T cells. Nature 372:691.[Medline]
24+ CD4-CD8- T cells. J. Exp. Med. 186:109. chain is used by a unique subset of major histocompatibility complex class I-specific CD4+ and CD4-8- T cells in mice and humans. J. Exp. Med. 180:1097. 24J Q antigen receptor in patients with systemic sclerosis. J. Exp. Med. 182:1163.ß T cells and endogenous IL-4 in non-MHC-restricted rejection of embryonal carcinoma in genetically resistant mice. J. Immunol. 158:5338.[Abstract]
24-J Q/V ß 11 T cell receptor is expressed in all individuals by clonally expanded CD4-8- T cells. J. Exp. Med. 180:1171./ß T cells demonstrates preferential use of several V ß genes and an invariant TCR chain. J. Exp. Med. 178:1.ß T cells expressing invariant TCR -chains. J. Immunol. 158:5603.[Abstract]
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S. Tangri, L. Brossay, N. Burdin, D. J. Lee, M. Corr, and M. Kronenberg Presentation of peptide antigens by mouse CD1 requires endosomal localization and protein antigen processing PNAS, November 24, 1998; 95(24): 14314 - 14319. [Abstract] [Full Text] [PDF]
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L. Brossay, O. Naidenko, N. Burdin, J. Matsuda, T. Sakai, and M. Kronenberg Cutting Edge: Structural Requirements for Galactosylceramide Recognition by CD1-Restricted NK T Cells J. Immunol., November 15, 1998; 161(10): 5124 - 5128. [Abstract] [Full Text] [PDF]
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L. Brossay, M. Chioda, N. Burdin, Y. Koezuka, G. Casorati, P. Dellabona, and M. Kronenberg CD1d-mediated Recognition of an {alpha}-Galactosylceramide by Natural Killer T Cells Is Highly Conserved through Mammalian Evolution J. Exp. Med., October 19, 1998; 188(8): 1521 - 1528. [Abstract] [Full Text] [PDF]
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J. L. Matsuda, L. Gapin, N. Fazilleau, K. Warren, O. V. Naidenko, and M. Kronenberg Natural killer T cells reactive to a single glycolipid exhibit a highly diverse T cell receptor beta repertoire and small clone size PNAS, October 23, 2001; 98(22): 12636 - 12641. [Abstract] [Full Text] [PDF]
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