By
From the * Laboratory of Immunology, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya
464, Japan; Department of Dermatology, Nagoya City University School of Medicine, Mizuho-ku,
Nagoya 467, Japan; and § Department of Chemical Hygiene and Nutrition, Faculty of Pharmaceutical
Sciences, Nagoya City University, Mizuho-ku, Nagoya 467, Japan
To elucidate the function of the mouse TL antigen in the thymus, we have derived two TL
transgenic mouse strains by introducing Tlaa-3 of A strain origin with its own promoter onto a
C3H background with no expression of TL in the thymus. These transgenic mouse strains,
both of which express high levels of Tlaa-3-TL antigen in their thymus, were analyzed for their T
cell function with emphasis on cytotoxic T lymphocyte (CTL) generation. A T cell response
against TL was induced in Tg.Tlaa-3-1, Tg.Tlaa-3-2, and control C3H mice by skin grafts from
H-2Kb/T3b transgenic mice, Tg.Con.3-1, expressing T3b-TL ubiquitously. Spleen cells from
mice that had rejected the T3b-TL positive skin grafts were restimulated in vitro with
Tg.Con.3-1 irradiated spleen cells. In mixed lymphocyte cultures (MLC), approximately 20%
and 15% of Thy-1+ T cells derived from Tg.Tlaa-3-1 and Tg.Tlaa-3-2, respectively, expressed
TCR, whereas almost all those from C3H expressed TCR
. The MLC from Tg.Tlaa-3-2
and C3H demonstrated high CTL activity against TL, while those from Tg.Tlaa-3-1 had little
or none. The generation of
CTL recognizing TL in Tg.Tlaa-3-2, but not C3H mice, was
confirmed by the establishment of CTL clones. A total of 14
CTL clones were established
from Tg.Tlaa-3-2, whereas none were obtained from C3H. Of the 14
CTL clones, 8 were
CD8+ and 6 were CD4
CD8
double negative. The CTL activity of all these clones was TL specific and inhibited by anti-TL, but not by anti-H-2 antibodies, demonstrating that they recognize TL directly without antigen presentation by H-2. The CTL activity was blocked by antibodies to TCR
and CD3, and also by antibodies to CD8
and CD8
in CD8+ clones,
showing that the activity was mediated by TCR
and coreceptors. The thymic origin of these
CTL clones was indicated by the expression of Thy-1 and Ly-1 (CD5), and also CD8
heterodimers in CD8+ clones on their surfaces and by the usage of TCR V
4 chains in 12 of
the 14 clones. Taken together, these results suggest that Tlaa-3-TL antigen expressed in the
thymus engages in positive selection of a sizable population of
T cells.
Mice have at least 30-40 genes called "nonclassical"
MHC class I or MHC class "Ib" genes mainly mapping to three chromosomal regions within the murine
MHC: H-2Q, T, and M (1, 2). Recent studies have demonstrated that some of the proteins encoded by these genes
are related to the classical MHC class I molecules in structure and in function, presenting antigens to TL antigens belong to the family of nonclassical MHC
class I antigens (6). At least one gene coding for a TL antigen is present in all mouse strains so far tested, and two or
more have been found in some strains; T3b has been found
in C57BL/6 (B6)1, T3k in C3H/He (C3H), T3d and T18d
in BALB/c and Tlaa-1, Tlaa-2 and Tlaa-3 in A strain. What
makes TL distinct from other MHC class I antigens is their
unique expression profile (6). TL antigens are expressed
in epithelial cells of the small and large intestines in all
mouse strains. Certain strains such as A and BALB/c also express TL on normal thymocytes, while others such as B6
and C3H do not. Even in B6 and C3H mice, however,
malignant transformation of T cell causes TL to be expressed.
The structures of TL genes and products are very similar
to those of classical MHC class I genes and products (6).
However, until our recent study, TL had not been shown
to behave like MHC class I antigens in terms of histocompatibility antigen. We derived H-2Kb/T3b transgenic mice
expressing the TL antigen ubiquitously under the action of
an H-2Kb promoter (11). By transplanting their skin onto
C3H mice, the background strain, we demonstrated that
TL can serve as a transplantation antigen and elicit TCR We have derived another pair of transgenic mouse strains
with Tlaa-3 of A strain origin. One of these, Tg.Tlaa-3-1,
expresses large amounts of TL antigen on thymocytes and has a small thymus consisting mainly of L3T4(CD4) TL Genes.
Three TL genes, T3b of B6 (14), Tlaa-3 of A strain
(15), and T3k of C3H (EMBL/GenBank/DDBJ accession No.
D86082) were involved in this study. The DNA sequences of
their coding regions and deduced amino acid sequences are very
similar to each other and >90% of their nucleotides and amino
acids are identical. Furthermore, all three TL molecules have
identical amino acid sequences in the putative peptide binding region (PBR) which determines the binding specificity of MHC
class I molecules to antigenic peptides and to TCR. Outside
PBR, however, there are differences as shown in Fig. 1.
Mice.
The derivation of transgenic mouse strains has been described previously (11). Briefly, a pair of transgenic strains,
Tg.Con.3-1 and Tg.Con.3-2, were produced by introducing a
chimeric gene consisting of the H-2Kb promoter region and exon
1 (encoding the signal peptide) and the T3b exons 2-6 (encoding
the mature protein). These mice express T3b-TL in almost all tissues including their skins and spleens. The other pair, Tg.Tlaa-3-1
and Tg.Tlaa-3-2, having a Tlaa-3 transgene with its own promoter from A strain, express Tlaa-3-TL predominantly on thymocytes. All transgenic mice were generated on a C3H background
which does not express TL in the thymus. C3H mice were purchased from Japan SLC Inc. (Hamamatsu, Shizuoka, Japan).
Antibodies.
The following mAbs and one conventional antibody (cAb) were developed in our laboratory, provided by various scientists, or purchased: Rat mAb to Lyt-2 (53-6.7; Dr. N. Shinohara, Mitsubishi Kasei Institute for Life Science, Machida,
Japan [16]), rat mAb to L3T4 (GK1.5; Dr. N. Shinohara [17]),
hamster mAb to TCR Flow Cytometric Analysis.
Flow cytometric analysis was performed with a FACScan®. For secondary reagents, PE-conjugated
streptavidin (Biomeda Corp., Foster City, CA), FITC-labeled anti-
hamster Ig (CALTAG Labs., South San Francisco, CA), anti-rat
Ig (Tago Inc., Burlingame, CA), or anti-mouse Ig (DAKOPATTS, Glostrup, Denmark) were used.
Skin Grafts.
Female Tg.Tlaa-3-1, Tg.Tlaa-3-2 and C3H mice
(6-10-wk-old) received full-thickness sections of skin (1-cm
disks) from the abdomens of Tg.Con.3-1 mice onto their backs.
Plaster casts were removed on day 10.
Establishment and Maintenance of CTL Bulk Cultures and Clones.
The methods for induction of CTL and assays for their cytotoxic
activity have been described previously (12). Briefly, 4-8 wk after
the rejection of grafted skin, spleen cells (5 × 107) from the recipient mice were cultured with 5 × 107 irradiated (2,000 R)
Tg.Con.3-1 spleen cells for 5 d. The cells were used as effector
cells in CTL assays and maintained as CTL bulk cultures by
weekly restimulation with irradiated Tg.Con.3-1 spleen cells in
the presence of human recombinant IL-2 (Takeda Chemical Industries Ltd., Osaka, Japan) at a concentration of 5 ng/ml. For
CTL assays, 51Cr-labeled target cells (2 × 104) were incubated
with various numbers of effector cells. For the blocking test of
CTL activity by antibodies, serially diluted antibodies were added
to mixtures of effector cells and labeled target cells. After incubation for 3 h at 37°C, the supernatants were harvested using a Supernatant Collection System (Skatron Inc., Sterling, VA) for measurement of 51Cr release. The percent specific lysis was calculated
using the following equation: 100 × (a or
T cells
(reviewed in reference 2), while others have completely different functions such as that acting as an Fc receptor for IgG (FcRn) (3). The idea that some nonclassical class I
molecules might function as restriction elements for
T cells
has been suggested by the distinctive tissue distribution and
limited V gene usage of
T cells. Indeed, several studies
have shown that
T cells recognize nonclassical class I
molecules encoded by genes located within the Q or T regions (reviewed in references 4, 5).
CD8+ CTL response (12). Furthermore, we showed that
this CTL response is TL restricted so that CTL directly recognizes TL or a complex of TL plus antigenic molecules
without any requirement for antigen presentation by classical MHC class I molecules. The results suggested the possibility that TL can serve as an antigen presenting molecule, similar to classical MHC class I antigens.
Lyt2(CD8)
double negative (DN) cells of
lineage, and it
develops a high incidence of T cell lymphomas, suggesting
that TL plays a critical role in T cell development in the
thymus, especially of the
T cell lineage (13). However,
the other Tlaa-3 transgenic strain, Tg.Tlaa-3-2, demonstrates no obvious abnormalities in thymic development and does not develop T cell lymphomas (11). In the
present study, to define the role of TL antigens expressed in
the thymus, we generated and characterized TL-restricted
CTL responses in Tg.Tlaa-3-1 and Tg.Tlaa-3-2 mice and
made a comparison with those in C3H strain.
Fig. 1.
Comparison of the predicted amino acid sequences of TL
molecules of Tlaa-3 of A mice, T3b of B6, and T3k of C3H. Only amino
acid residues different among the three TL molecules are shown. Amino
acid sequences are given by the single-letter code, and amino acid numbers in the mature peptide are noted above the sequences.
[View Larger Version of this Image (24K GIF file)]
(H57-597; Dr. R.T. Kubo, National
Jewish Center for Immunology and Respiratory Medicine, Denver, CO [18]), hamster mAb to TCR
(3A10; Dr. S. Tonegawa,
Massachusetts Institute of Technology, Cambridge, MA [19]),
hamster mAb to TCR
(GL3; Cedarlane Labs. Ltd., Hornby, Ontario, Canada), hamster mAb to V
4 (UC3-10A6; PharMingen, San Diego, CA), hamster mAb to CD3 (145-2C11; Dr. J.A.
Bluestone, The University of Chicago, Chicago, IL [20]), mouse
mAb to TL.2 (TT213 [12]), mouse mAb to Ly-1.1, mouse mAb
to Lyt-3.2 (ID9P35), rat mAb to Thy-1.2 (Becton Dickinson,
Immunocytometry Sys., Mountain View, CA), and cAb to H-2k
([B6 × DBA/2] anti-C3H mammary tumor, MM48).
b/c
b), where a is the
radioactivity in the supernatant of target cells mixed with effector
cells, b is the radioactivity in the supernatant of target cells incubated without effector cells, and c is the radioactivity in the supernatant after complete lysis of target cells with 2% Triton X-100.
Target Cells.
Con A-stimulated spleen cells of Tg.Con.3-1
and C3H were prepared as previously described (12). In addition
to Con A-stimulated spleen cells, the following cells were used as
targets for CTL assays, with the amounts of TL gene product on cell
surfaces were estimated by flow cytometry with TT213 anti-TL
mAb: Con A blasts of Tg.Con.3-1 (mean fluorescence intensity
[MFI] of stained cells - MFI of unstained cells; 298.6, expressing
T3b), C3H (0.1, TL) and B6 (1.5, TL
), thymidine kinase-negative L-cells ([Ltk
], 4.0, TL
), and H-2Kb/T3b Ltk
transfectants (785.0, T3b), as well as ERLD (derived from B6, 253.5, T3b), RADA1 (derived from A strain, 178.9, Tlaa-1, -2, and -3),
and ASL-1 (derived from A strain, 584.1, Tlaa-1, -2, and -3) leukemias maintained in vivo.
RNA Extraction and Reverse Transcription-PCR.
Total RNA
was extracted from CTL clones using TRIzol Reagent (GIBCO
BRL, Gaitherburg, MD). 10 µg of total RNA were reverse transcribed into first strand cDNA with 3 µg of random primers and
1,000 U of SuperScript II reverse transcriptase (RT) (GIBCO BRL). TCR chains were amplified using V
-specific primers
and a single common C
primer by the methods of Takagaki
(21). The nomenclature of V
is according to Reilly et al. (22).
The RT-PCR products were separated by agarose gel electrophoresis.
Nucleotide Sequencing of the V-J
Junction.
PCR products were
cloned into a T-tailed M13mp18 vector and sequenced by a cycle
sequencing method using an automated DNA sequencer (model
373A; Applied Biosystems, Inc., Foster City, CA).
To investigate the role of TL antigen expressed in the thymus, a T
cell response against TL was induced by transplanting T3bTL positive skin of Tg.Con.3-1 onto Tg.Tlaa-3-1 and
Tg.Tlaa-3-2 mice, two transgenic strains expressing Tlaa3-TL in the thymus, and also onto C3H mice without thymic TL expression (Fig. 2). All three strains rejected the
grafted skin within 3 wk, in accordance with our previous
data (12). The spleen cells from mice which had rejected
Tg.Con.3-1 skin were restimulated in vitro with irradiated
T3b-TL+ spleen cells from Tg.Con.3-1. Spleen cells of
Tg.Tlaa-3-2 and C3H reacted vigorously and about 80%
were found to be blastic cells expressing Thy-1 and CD3
on their surfaces. In contrast, Tg.Tlaa-3-1 reacted weakly
and only 25% were Thy-1+CD3+ blastic cells. (Fig. 3 A).
Of the Thy-1+ T cells, almost all of those derived from
C3H expressed TCR, while ~15% from Tg.Tlaa-3-2
and >20% from Tg.Tlaa-3-1 expressed TCR
. In the
T cell population derived from Tg.Tlaa-3-2, two thirds
were L3T4
Lyt-2+, while approximately one third were
L3T4
Lyt-2
double negative (DN), and very few were
L3T4+Lyt-2
(Fig. 3 B). Of the
T cells from Tg.Tlaa-3-1,
~60% were DN, 25% were L3T4
Lyt-2+, and 15% were
L3T4+Lyt-2
(data not shown). In the
T cell population derived from both Tg.Tlaa-3-2 and C3H, >70% were
L3T4
Lyt-2+ and the rest were L3T4+Lyt-2
. In contrast,
the
T cell population from Tg.Tlaa-3-1 consisted of
~45% L3T4
Lyt-2+, 45% L3T4+Lyt-2
and 10% DN
cells (data not shown).
The in vitro stimulated T cells were tested for cytotoxic
activity against TL+ targets. T cells from Tg.Tlaa-3-2 and
C3H showed high levels of CTL activity against TL+ target
cells, but not TL cells (Fig. 4 A). Tg.Tlaa-3-1, however,
yielded little or no CTL activity (data not shown), confirming the previous finding that the T cell function of
Tg.Tlaa-3-1 is severely impaired (13). Therefore, in the
subsequent studies, only CTL from Tg.Tlaa-3-2 and C3H
were characterized. The CTL activity of Tg.Tlaa-3-2
against various target cells was not different from that of
C3H which has been previously described (12). As shown
in Fig. 4 B, the CTL activity from both was blocked by
anti-TL antibody, but not by anti-H-2k, thus confirming
that CTL recognizes TL directly without involving antigenic presentation by H-2 (12). The CTL activity of C3H was also blocked by anti-TCR
, CD3, and Lyt-2 antibodies but not by anti-TCR
or anti-L3T4. In the
Tg.Tlaa-3-2 case, the CTL activity was blocked partially by
anti-TCR
antibody, although not as strongly as shown
by anti-TCR
antibody, indicating a certain population
of
T cells. Together with the data from flow cytometric
analysis, these results suggested that a small but significant
proportion of CTL recognizing T3b-TL was skewed toward the
lineage by the presence of Tlaa-3-TL antigen
in the thymus.
Establishment of
To examine further the
influence of TL antigen expressed in the thymus on the
CTL population, TL-reactive CTL clones were established
from Tg.Tlaa-3-2 and C3H. 23 clones were initially derived from four Tg.Tlaa-3-2 mice and the same number
from three C3H mice. By flow cytometric and antibody
blocking analyses, 3 out of the 23 CTL clones derived from
Tg.Tlaa-3-2 mice were found to express TCR, whereas
all C3H CTL clones expressed TCR
. Thus, establishment of the CTL clones indicated that
CTL are induced
characteristically in mice expressing TL in their thymus,
confirming the results of bulk cultures.
The three CTL
clones described above were all Lyt-2+ (see below), although one third of
T cells in bulk culture were DN as
shown in Fig. 3 B. To test whether TL-reactive
CTL
clones with DN phenotype could be established, 11 additional
CTL clones were derived from five Tg.Tlaa-3-2
mice. Thus, a total of 14
CTL clones were characterized, as summarized in Table 1. Flow cytometric analysis
showed eight to be Lyt-2+ and six to be DN. Lyt-2 is a
CD8
chain, and in T cells of thymic origin, it forms a
heterodimer with Lyt-3, a CD8
chain, while it forms an
homodimer in those of intestinal epithelium (23). All
Lyt-2+
CTL clones established in this study also expressed Lyt-3, indicating a thymic origin. In addition, a
thymic origin of not only Lyt-2+, but also DN
CTL
clones, was indicated by the fact that all clones expressed
Thy-1 and Ly-1 (CD5) on their cell surface (Table 1). Both
antigens have been shown to be expressed on
T cells of thymic origin, but not on those of extra-thymic origin
(24, 25).
|
The 14 clones were examined for their cytotoxic activity against TL+ and TL
target cells. All lysed TL+ target
cells including Con A blasts of Tg.Con.3-1 and RADA1, but not TL
cells including Con A blasts of C3H and B6.
The results with CTL clones were essentially identical to
those with the bulk culture. The
CTL clones were also
tested by antibody blocking for their target specificity and
the expression of TCR and coreceptor molecules. The results are summarized in Table 1 and representative findings are shown in Fig. 5. Fig. 5, A and C, illustrate results for one of the Lyt-2+
CTL clones, KC4-4, and Fig. 5, B and D,
for one of the DN
CTL clones, KC8-3. The CTL activity of all these
CTL clones was blocked by anti-TL
antibodies, indicating the TL-specific CTL activity. Blocking
by anti-TL antibodies but not by anti-H-2k antibody further indicated that these
CTL clones directly recognize
the TL molecule. The CTL activity was also blocked by anti-TCR
and CD3 antibodies. All Lyt-2+
CTL
clones were blocked by anti-Lyt-2 and anti-Lyt-3 antibodies while all DN clones were not, confirming the results of
flow cytometric analysis. Other than the Lyt-2 and Lyt-3
expression, these 14
CTL clones showed no differences
in their expression of adhesion, costimulatory, and accessory molecules; all
CTL clones were positive for Pgp-1
(CD44), LFA-1 (CD11a), ICAM-1 (CD54), CD2, CD28,
CTLA-4, and IL-2R
/
, and negative for L-selectin
(CD62L), VLA-4
(CD49d), and HSA (data not shown).
To further confirm the thymic origin of these CTL
clones, their V
segment usage was determined. By RTPCR, nine clones yielded V
2 and V
4 products, two
clones V
1 and V
2, two clones V
1, V
2 and V
4, and
one clone V
4, while no clones yielded V
5, V
6, or V
7
products (Table 1). There was no distinctive difference in
the usage of V
by Lyt-2+ clones from that by DN clones.
All of 12 clones with RT-PCR V
4 products reacted with
an antibody specific to V
4 in the flow cytometric analysis,
indicating that V
4 chains were indeed expressed on the
cell surface. It has been reported that the V
4 chain is preferentially used by thymus-dependent
T cells, while V
1 and V
2 chains are used by both thymus-dependent and
-independent
T cells (26, 27). Thus, the results indicated that most, if not all, of these clones were derived from
adult thymus. To determine the diversity in the CDR3
region of
chains, RT-PCR products from seven representative clones, three Lyt-2+ and four DN, were sequenced
(Fig. 6). One or two in-frame products were identified and
they contained N region nucleotides at all the V-J junctions except for V
2 and V
4 chains in KC1-3 and KC4-8, respectively. The deduced amino acid sequences for the
CDR3 were different from one another except for those of
the V
2 chains in the KC6-1 and KC7-4 clones, suggesting that a diverse set of
TCR recognize the TL molecule. In summary, the usage of V
4 chains with N sequences in 12 of 14 clones, and the expression of Thy-1,
Ly-1, and CD8
heterodimer in the case of Lyt-2+ clones,
indicated that these
CTL clones that recognize TL were
matured in adult thymus.
The present study on the role of TL antigen expressed in
the thymus on T cell selection demonstrated that a sizable
population of CTL recognizing T3b-TL induced in Tlaa-3-2
transgenic mice are T cells, in contrast to those in C3H mice which were found to be mostly
T cells. Most of
the
CTL were of adult thymic origin; all of them expressed Thy-1, Ly-1 (CD5) and CD8
heterodimers in
the cases of Lyt-2+ clones, and 12 of 14 clones used TCR
V
4 chains with N sequences in the V-J junctions except
for V
4 of KC4-8. Thus, our results indicate that the presence of Tlaa-3-TL in the thymus promotes the positive selection of
T cells recognizing T3b-TL. T3b and Tlaa-3
have identical amino acids in the putative PBR which determine the binding specificity to antigenic peptides and to
TCR. Therefore, it is most likely that
T cells, selected
on the basis of their affinity for Tlaa-3, are able to react
with T3b as well, and that such
T cells are stimulated to
generate CTL against T3b by T3b+ skin grafts. Thus, the
presence of TL molecules in the thymus is required for the
selection of
T cells reactive to TL, and cannot be substituted for by any other MHC class I or CD1 molecules expressed in C3H mice. This notion is supported by the expansion of
T cells in the thymus and periphery of the
other transgenic mouse strain, Tg.Tlaa-3-1, as we reported
previously (13). An association of TL antigen with
T cells
has also been speculated from the observation that it is expressed in the intestinal epithelium and
T cells are
present nearby (9, 10), although no direct evidence for any
interaction between the intestinal epithelium and
T cells has been presented.
It is an intriguing question whether normal mice that express TL in the thymus also produce CTL with specificity for TL. However, they do have at least two, and as
many as four, TL genes (6, 31) which are expressed simultaneously in the thymus at levels which differ from strain to
strain. Therefore, it will be a very difficult task to determine which TL genes are responsible for T3b-specific responses and for selection of
CTL. Although two TL
congenic strains, B6-TL+ and A-TL
are available, it is
known that they are different from their counterparts, B6
and A, not only in terms of their TL genes but also for as
many as 20 other MHC class I genes located in the TL region (32). Some of these class I genes have very similar structures and expression patterns to TL genes (10, 32), and some have been shown to cause skin graft rejection (33).
Accordingly, it will be very difficult to rule out their involvement in TL recognition. To unravel the complexity
of the TL system, we have generated and analyzed transgenic mice with Tlaa-3 gene driven by its own promoter as
a first step. A definite answer can only be obtained from the
analysis of transgenic mice with other TL genes, such as
Tlaa-1, Tlaa-2 and T18d, which are currently under construction.
Regarding the specificity, the finding that both bulk and
cloned CTL were cytotoxic against Tlaa-3-expressing
RADA1 suggested that they may be auto-reactive. RADA1,
however, expresses at least two other TL genes, in addition to Tlaa-3 (6). Thus, there remains a possibility that the reactivity is directed against antigens encoded by TL genes
other than Tlaa-3. To clarify this point, target cells expressing only Tlaa-3 are needed. Transfection experiments to
this end are now underway. Only after they are concluded
and modes of recognition of TL molecules by CTL are
elucidated (see below), will we be in a position to answer
the questions of whether and how the negative selection operates against Tlaa-3 in the thymus.
The majority of T cells in mice and humans and most
T cell clones or hybridomas reported so far do not express either CD4 or CD8 (4, 5). This suggests that
T cells
recognize MHC or non-MHC molecules with low or no
affinity to CD4 or CD8 coreceptors, or that the maturation
and activation of
T cells do not require the presence of
CD4 or CD8 molecules. Since there have hitherto been
only few reports of
T cell clones with clear CTL or
helper activity, it has been difficult to conduct detailed studies on the requirement of coreceptors for
T cells.
Our present finding that 8 of 14 TL-restricted
CTL
clones express CD8
, supports the proposal that TL can
bind CD8 (34) and further implies that the CD8-p56lck signal pathway can operate in
CTL, similarly to the
CTL case (35). In addition to CD8+
CTL, DN
CTL
clones were also established. Analysis of DN
CTL clones
has so far shown no differences from CD8+
CTL clones
in their cytotoxic specificity or activity, or in the expression of costimulatory or adhesion molecules except CD8.
The differential expression of CD8 in these
CTL may
be due to the affinity of TCR for the target, or to the density of antigenic epitopes. Measurement of the affinity of
TCR
expressed in CD8+ and DN CTL clones, and detailed analysis of the target molecules of these
CTL, are
now needed to clarify these issues.
Most of our sequenced CTL clones were found to
have two in-frame TCR
chain transcripts. Further studies with transgenic mice are needed to determine which inframe
chain is responsible for TL recognition, although
surface expression of the V
2 chain is in doubt (30, 36). It
has been speculated that the TL molecule, a member of the
nonpolymorphic MHC class I molecules, may be recognized by a limited set of TCR (37). However, this seems to
be unlikely since the present analysis showed diversity in
the CDR3 region of
chains, although analysis of
chains is
still necessary. The results suggest heterogeneity in antigenic
epitopes and TL recognition by CTL as discussed below.
Tlaa-3, T3k, and T3b have identical amino acid sequences in their PBRs. In their whole proteins, however,
Tlaa-3 and T3b differ at over 30 amino acid positions, while
T3k and T3b are different at a single amino acid position,
276 in the transmembrane region (Fig. 1). T3k expressed in
the intestine of C3H and Tlaa-3 transgenic mice may not
have any significance for the recognition of T3b, since a
previous experiment suggested that T3b expressed in the
intestine does not induce tolerance against T3b itself expressed in the skin (12). Identification of the antigenic epitopes recognized by and
CTL against TL should
reveal antigenic determinant(s) responsible for skin graft rejection. The CTL against TL were all TL-restricted, regardless of the C3H or Tg.Tlaa-3-2 origin and usage of
TCR
or TCR
. There are several possible modes in
which CTL can recognize TL molecules. For example,
CTL may recognize a complex of TL and endogenous
peptides, similar to conventional CTL recognition of classical MHC molecules (38), or a complex of TL and nonpeptide antigens such as CD1 plus lipids (39). CTL may interact with TL molecules without any contribution from
bound peptides, possibly from the side of the TL molecule, away from the putative PBR of the
1 and
2 domains
(40), or with a complex of TL molecules and peptides derived from TL itself. The CTL may be heterogeneous in
TL recognition; some CTL may recognize TL + peptides
while others may recognize TL itself. To determine how
TL molecules are recognized by CTL, the established CTL
clones will be tested further on various TL+ target cells including transfectants of Drosophila melanogaster cells expressing TL molecules devoid of binding endogenous peptides (41). Recent studies using a TAP-2-deficient cell line,
RMA-S, have demonstrated that the transport of TL molecules to the cell surface is TAP-2 independent (31, 42), but
these findings do not necessarily exclude the possibility that
TL molecules bind antigenic peptides by other mechanisms. Our previous structural comparison of TL antigens
with classical class I antigens suggested that TL molecules
can form peptide-binding clefts (37). There has been a report that they bind to peptides modified at the amino termini (43), although an absence of peptides bound to TL has been also reported (42). The CTL assay is by far the most
sensitive method for detecting antigenic peptides bound to
MHC molecules (44), and its application should allow
more complete elucidation of the function of TL in the
immune system.
Address correspondence to Yuichi Obata, Laboratory of Immunology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya 464, Japan.
Received for publication 24 June 1996
We are indebted to Dr. L.J. Old for his continuous encouragement and advice thoughout these studies. We thank Y. Matsudaira, S. Ozeki, and H. Tamaki for their excellent technical assistance. We thank Dr. K. Furukawa, Dr. K. Kuribayashi, Dr. M. Miyasaka, Dr. M. Muto, Dr. E. Nakayama, Dr. S. Sakaguchi, and Dr. H. Yagita for their kind gifts of monoclonal antibodies.This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas and a Grantin-Aid for General Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan, and a Grant-in-Aid from the Ministry of Health and Welfare, Japan. This work was also supported in part by a grant from Naito Foundation and a grant from Imanaga Foundation.
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