Thymus leukemia antigen (TL)-specific cytotoxic T lymphocytes recognize the {alpha}1/{alpha}2 domain of TL free from antigenic peptides

Kunio Tsujimura1, Yuichi Obata3, Eisei Kondo1, Keiko Nishida1, Yasue Matsudaira1, Yoshiki Akatsuka1, Kiyotaka Kuzushima1 and Toshitada Takahashi1,2

1 Division of Immunology and 2 Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan 3 Department of Biological Systems, RIKEN BioResource Center, Tsukuba Institute, RIKEN, 3-1-1 Koyadai, Tsukuba 305-0074, Japan

Correspondence to: K. Tsujimura; E-mail: ktsujimu{at}aichi-cc.jp
Transmitting editor: K. Okumura


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The thymus leukemia antigens (TL) belong to the MHC class Ib family and can be recognized by CD8-dependent or -independent cytotoxic T lymphocytes (CTL) showing TL, but not H-2, restriction. We previously reported that the CTL epitope is TAP independent and in the present study we further characterize the recognition mechanism of CD8-dependent TL-specific TCR{alpha}ß CTL. We first prepared empty TL tetramers by way of peptide-independent folding with recombinant proteins produced in an Escherichia coli expression system, and showed that TL-specific CTL recognized TL without putative TL-associated peptide and/or post-translational modifications of TL by mammalian and insect cells. We next prepared transfectants expressing various chimeric TL molecules with mouse or human MHC class I as well as chimeric TL tetramers with recombinant proteins produced by insect cells, and demonstrated that chimeric TL whose {alpha}3 domain was replaced by that of H-2Kb, but not of HLA-A2, was sufficient for binding and activation of TL-specific CTL. These results indicate that TL-specific CTL recognize predominantly their {alpha}1/{alpha}2 domain as an epitope(s) and that the binding activity to the murine CD8 of the {alpha}3 domain of H-2Kb is sufficient to induce their CTL activity, although it is known to be weaker than that of TL, but stronger than that of HLA. The results taken together indicate that CD8-dependent TL-specific TCR{alpha}ß CTL recognize an epitope(s) of the {alpha}1/{alpha}2 domain of TL free from antigenic molecules, and that CD8 plays an important role in stable interactions between TL and their corresponding TCR.

Keywords: CD8, cytotoxic T lymphocyte, MHC class Ib, TCR, thymus leukemia antigen


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The mouse thymus leukemia antigens (TL) belong to the MHC class Ib family and their expression is restricted to the intestines in all mouse strains as well as to the thymus of TL+ strains (e.g. A-strain and BALB/c mice) (14). Recent studies by ourselves and others have revealed that TL tetramers can bind to intestinal intraepithelial lymphocytes (iIEL) and thymocytes in a CD8-dependent, but TCR/CD3-independent, manner, while their binding to TL-specific CTL is TCR/CD3 and CD8 dependent (5,6). Possible roles of TL in the intestines (6) and the thymus (7) have been suggested.

We have shown that TL can be recognized directly by both TCR{alpha}ß and TCR{gamma}{delta} CTL without any requirement for antigen presentation by H-2 molecules (79), and there is now evidence that TL molecules are transported to and stably expressed on the surfaces of TAP-deficient cells (1013). We have further demonstrated that most TL-specific CTL recognize an epitope(s) expressed on TL in a TAP-independent manner (10). Very recently, two reports have indicated that T18d-TL has no antigen-binding groove wide enough to accommodate a peptidic or lipid antigen (14) and that it can be refolded peptide-independently at least in vitro (15). Although T18d-TL tetramers generated without peptides can bind to CD8{alpha}{alpha} homodimers expressed on iIEL with the {alpha}3 domains (15), it remains to be determined whether TL-specific CTL can recognize TL molecules free from antigenic peptides.

We previously reported cytotoxic T lymphocyte (CTL) activity against TL through the perforin/granzyme pathway to be strongly dependent on the TL–CD8 interaction (9). Since the binding affinity of TL for CD8 is known to be higher than that of other MHC class I molecules (6,16), it is an interesting question whether this might be in fact necessary for the activation of TL-specific CTL, as in the case of the TL–CD8 interaction observed in the small intestines and the thymus (5,6). Since these CTL are considered to be self-reactive, the binding affinity of their TCR to TL can be speculated to be relatively low, suggesting an importance of TL–CD8 interactions for activating TL-specific CTL.

In the present study, empty TL tetramers were generated first by way of peptide-independent folding (15) to ascertain whether the recognition of TL-specific TCR{alpha}ß CTL is independent of putative TL-associated peptides and/or post-translational modifications of TL by the mammalian and/or insect cells used as the target cells. Next, various chimeric TL molecules were prepared with the {alpha}3 domain of T3b-TL replaced by those of mouse or human MHC class I to allow analysis of the epitope(s) for TL-specific CTL and their CD8 dependence in TL recognition. Finally, the CDR3 lengths of {alpha} and ß TCR polypeptide chains were analyzed to determine the characteristics of {alpha}ß TCR expressed on TL-specific CTL. Through the analyses described above, we attempted to elucidate the recognition mode of TL-specific CTL and their CD8-dependence, and demonstrated that TL-specific CTL recognize the {alpha}1/{alpha}2 domain of TL without antigenic peptides.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cells
CTL clones against TL or H-2Kb were established as previously described (710). All six TL-specific CTL clones used for the present study express TCR{alpha}ß and show CD8-dependent cytotoxicity (9). iIEL were prepared from C3H/He mice (Japan SLC, Hamamatsu, Japan) as described earlier (17).

Antibodies
Rat mAb against TL [HD168 and HD177 (18)] and a mouse mAb to TL.2 [TT213 (8)] were described previously. A rat mAb against H-2KbDb [20-8-4S (19)] and a mouse mAb to HLA-A2 [MA2.1 (20)] were kindly provided by Dr E. Nakayama (Okayama University, Okayama, Japan) and Dr K. Itoh (Kurume University, Kurume, Japan) respectively.

Preparation of tetramers through peptide-independent folding
A cDNA encoding the truncated T3b [encoding from amino acids 1 to 279 (in the transmembrane domain)], followed by a GlySer linker (GS), a biotin-specific peptide (BSP), and a histidine tag (6xHis), was prepared by PCR using the construct for the baculovirus expression system described previously (5) as a template. NdeI and HindIII sites were introduced at the 5' and 3' ends respectively, and the PCR products were ligated into the pET-23a(+) vector (Novagen, Madison, WI). A cDNA encoding murine ß2-microglobulin without the leader sequence was also prepared by PCR, and ligated into the NdeI and HindIII sites of the pET-23a(+) vector. The proteins were expressed as inclusion bodies in Escherichia coli BL21(DE3)pLys (Novagen) after induction with 0.4 M isopropyl-ß-D-thiogalactopyranoside. Purification of the proteins and preparation of tetramers through peptide-independent folding were conducted as described (5,15,21).

Preparation of transfectants
Chimeric cDNAs of T3b and H-2Kb or HLA-A2 were prepared by introducing a BamHI site at the junction of the {alpha}2 and {alpha}3 domains by PCR, and inserted into EcoRI–XbaI sites of pcDNA3.1/Zeo(+) (Invitrogen, Carlsbad, CA). Human 293 cells were transfected with pcDNA3.1/Zeo(+) vectors containing various cDNAs using TransIt-293 (Mirus, Madison, WI) and stably transduced cells were selected with Zeocin (Invitrogen).

Preparation of tetramers in a baculovirus expression system
A cDNA encoding a truncated T3b (encoding from the leader sequence to amino acid 279)–GS–BSP–6xHis was prepared by PCR using the construct for the baculovirus expression system described previously (5) as a template, and EcoRI and XbaI sites were also introduced at the 5' and 3' ends respectively. A mouse ß2-microglobulin cDNA tagged with SmaI and KpnI sites was prepared by PCR. cDNAs encoding soluble T3b–GS–BSP–6xHis and mouse ß2-microglobulin were inserted into EcoRI–XbaI and SmaI–KpnI sites respectively of the pFASTBACDUAL plasmid vector (Invitrogen).

A chimeric cDNA of T3b and H-2Kb was prepared by introducing a BamHI site at the junction of the {alpha}2 and {alpha}3 domains by PCR, tagged with GS–BSP–6xHis, and inserted into EcoRI–XbaI sites of pFASTBACDUAL, together with murine ß2-microglobulin as described above. Chimeric cDNA of T3b and HLA class I (A2 or E) were also prepared in the same way, tagged with GS–BSP–6xHis, and inserted into EcoRI/XbaI sites of pFASTBACDUAL, together with human ß2-microglobulin. To prepare mutant T3b-TL tetramers with a weaker binding capacity to CD8 than wild-type T3b-TL, alanine at position 245 was substituted with valine (22) by site-directed mutagenesis using T3b–GS–BSP–6xHis (and murine ß2-microglobulin) in pFASTBACDUAL as a template and a QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA).

Baculoviruses producing T3b–GS–BSP–6xHis or its derivatives associated with murine or human ß2-microglobulin were prepared using the Bac-TO-Bac baculovirus expression system (Invitrogen) according to the manufacturer’s instructions. Soluble products were purified from the culture supernatant with Ni-NTA agarose (Qiagen, Tokyo, Japan) and further procedures for tetramer preparation were performed as described (5,21).

Flow cytometric analysis
Flow cytometric analysis was performed on a FACSCalibur (Becton Dickinson Immunocytometry Systems, Mountain View, CA). For secondary reagents, FITC-labeled goat anti-rat IgG (Chemicon, Temecula, CA) or rabbit anti-mouse Ig (Dakopatts, Glostrup, Denmark) were employed.

CTL and IFN-{gamma} production assays
CTL clones (1 x 105) were incubated with various target cells (1 x 105) for 18 h in 96-well round-bottom tissue culture plates and then the culture supernatants were collected for measurement of IFN-{gamma} amounts by ELISA (Quantikine M; R & D systems, Minneapolis, MN).

Analysis of the CDR3 length of CTL clones
TCR{alpha} and ß usage of CTL clones was determined by RT-PCR as described (23,24), and nucleotide sequences were analyzed on an ABI Prism 3100 genetic analyzer (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Then, CDR3 lengths of CTL clones were determined as described (24,25).


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
TL-specific TCR{alpha}ß CTL clones recognize TL free from binding peptides and post-translational modifications
We previously reported that all CD8-dependent TL-specific TCR{alpha}ß CTL clones established so far recognize an epitope expressed TAP independently in experiments using a TAP-deficient cell line RMA-S and an insect cell line S2 expressing TL as target cells (10). Recent studies have demonstrated that the antigen-binding groove of TL is too narrow to present a peptidic or lipid antigen (14) and that TL can have a good conformation even when refolded without peptides (15). However, it remains to be determined whether TL free from additional antigenic molecules can be recognized by TL-specific CTL. To address this issue, we prepared T3b-TL tetramers with recombinant proteins produced in an E. coli expression system and refolded without addition of any antigenic peptides, as reported by Weber et al. (15), and then their reactivity against CD8-dependent TL-specific TCR{alpha}ß CTL clones was tested. As shown in Fig. 1, T3b-TL tetramers refolded without peptides were found to stain TL-specific CTL clones and the fluorescence intensity was comparable to that with T3b-TL tetramers prepared with proteins produced by a baculovirus expression system (5). iIEL and thymocytes were also positively stained with newly prepared TL tetramers (data not shown), similarly to those prepared with the protein produced by insect cells. These results suggest that most TL-specific CTL recognize the framework of TL molecules, and that post-translational modifications by mammalian and/or insect cells are not involved in the antigenic determinant formation.



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Fig. 1. Binding activity of T3b-TL tetramers prepared by way of peptide-independent folding to TL-specific TCR{alpha}ß CTL clones. CTL clones (1 x 106) were incubated at 37°C for 30 min with the phycoerythrin-labeled T3b-TL tetramers (1 µg/ml) free from peptide prepared with the recombinant proteins expressed in an E. coli expression system, together with those prepared with the proteins produced by insect cells (5) and then analyzed on a FACSCalibur. Both CTL clones are stained with T3b-TL tetramers prepared in E. coli and in insect cells. Six TL-specific TCR{alpha}ß CTL clones listed in Table 1 were analyzed and similar results were obtained in all cases.

 
TL-specific TCR{alpha}ß CTL clones recognize the {alpha}1/{alpha}2 domain of TL as an epitope
To elucidate the epitope(s) of TL-specific CTL clones, we next established transfectants (human 293 cells) expressing various chimeric T3b-TL as shown in Fig. 2. Profiles of FACS analysis revealed that the chimeric molecules are well expressed and have good conformation at 37°C. Thus, the reactivity of six CD8-dependent TL-specific TCR{alpha}ß CTL clones was tested against these transfectants. As can be seen from the representative data shown in Fig. 3, all these CTL clones recognized a T3b/Kb chimeric molecule consisting of the {alpha}1/{alpha}2 domain of T3b-TL and the {alpha}3 domain of H-2Kb, and produced IFN-{gamma} as with the whole T3b-TL molecule, but they could not recognize Kb/T3b. In addition, they did not recognize T3b/A2 consisting of T3b-TL ({alpha}1/{alpha}2) and HLA-A2 ({alpha}3) or A2/T3b consisting of HLA-A2 ({alpha}1/{alpha}2) and T3b-TL ({alpha}3). These results altogether suggest that the CTL recognize predominantly the {alpha}1/{alpha}2 domain of T3b-TL, and that the {alpha}3 domain of H-2Kb, with weaker binding affinity to CD8 than that of TL (6,16), is sufficient for the binding and activation of TL-specific CTL.



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Fig. 2. Flow cytometric analysis of transfectants expressing various T3b-TL chimeric molecules. Stably transfected 293 cells with various cDNA constructs were indirectly stained with anti-TL, H-2Kb or HLA-A2 and analyzed on a FACSCalibur. All mAb used in this study were found to recognize predominantly the {alpha}1/{alpha}2 domain of their respective molecules. Since 293 cells used as the parental cells express HLA-A2 endogenously, positive staining was observed. The transfectants with A2/T3b chimeric DNA expressed a higher amount of HLA-A2 than the parental cells. The reason why the remaining transfectants were almost negative for HLA-A2 expression is not known. One possibility might be that endogenous ß2-microglobulin is consumed by overexpressed MHC class I molecules. Components of chimeric constructs are illustrated on the right side of the figure (see Methods).

 


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Fig. 3. Reactivity of TL-specific TCR{alpha}ß CTL clones against transfectants expressing various T3b-TL chimeric molecules. CTL clones (1 x 105) were incubated with different transfectants (human 293 cells) expressing various T3b-TL chimeras for 18 h and the IFN-{gamma} concentration in the supernatants was measured by ELISA. Six TL-specific TCR{alpha}ß CTL clones listed in Table 1 were analyzed. Only results for two clones are illustrated, but similar results were also obtained with the other four.

 
The most probable explanation for the non-reactivity of the TL-specific CTL against T3b/A2 is that murine CD8 has very weak binding affinity to the {alpha}3 domain of HLA class I (2628) so that it cannot assist these CTL clones to bind to the target cells stably. Accordingly, to test this possibility we prepared chimeric tetramers consisting of T3b-TL ({alpha}1/{alpha}2) and HLA-A2 or -E ({alpha}3) plus human ß2-microglobulin using a baculovirus expression system (TL/HLA chimeric tetramers). The molecular ratio of chimeric TL and ß2-microglobulin was found to be 1:1 by SDS–PAGE, and the complexes reacted well with anti-TL mAb (HD168, HD177 and TT213) when subjected to sandwich ELISA (data not shown), suggesting the conformation of these tetramers to be well conserved. As shown in Fig. 4, iIEL and thymocytes were not stained with TL/HLA chimeric tetramers, confirming that these do not have any capacity for binding to murine CD8, in contrast to TL tetramers. In addition, TL-specific CTL clones were also not stained with these tetramers, whereas they were with T3b/Kb tetramers. The reason for the loss of binding of CTL clones to TL/HLA chimeric tetramers seemed not due to the human ß2-microglobulin, since they were stained with T3b-TL tetramers assembled with human ß2-microglobulin (data not shown). These results altogether suggest that TL-specific TCR{alpha}ß CTL clones cannot bind to target cells without the help of CD8, even if they may recognize their corresponding epitope(s) on TL located in the {alpha}1/{alpha}2 domain.



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Fig. 4. Binding activity of mutated or chimeric T3b-TL tetramers to TL-specific CTL clones, iIEL and thymocytes. Cells (1 x 106) were incubated with the various phycoerythrin-labeled TL tetramers prepared with the protein produced by insect cells (1 µg/ml) at 37°C for 30 min and analyzed on a FACSCalibur. Six TL-specific TCR{alpha}ß CTL clones listed in Table 1 were analyzed. Only results for two clones are illustrated, but similar findings were also obtained with the other four. Components of mutated and chimeric tetramers are illustrated on the right side of the figure (see Methods).

 
An additional tetramer whose alanine at position 245 of T3b-TL was replaced by valine (T3b-245V) was also prepared, since this mutation is reported to diminish the binding of HLA-A0201 tetramers to CD8 (22). Interestingly, T3b-245V as well as T3b/Kb tetramers exhibited greatly diminished binding to iIEL, whereas that to thymocytes still remained. These results confirmed that {alpha}3 domains of TL bind to CD8{alpha}{alpha} homodimers on iIEL as well as to CD8{alpha}ß heterodimers on thymocytes (5,6) and suggested that their modes of binding are quite different.

The CDR3 lengths of {alpha} and ß TCR polypeptides of TL-specific TCR{alpha}ß CTL clones are nearly identical
Most TCR{alpha}ß T cells recognize MHC plus antigenic peptides using {alpha} and ß TCR polypeptide chains whose CDR3 lengths are nearly identical (25). In contrast, when most TCR{gamma}{delta} T cells and Ig recognize their respective epitopes, the CDR3 lengths of {delta} and H chains are reported to be longer than those of their partners, {gamma} and L chains respectively (25). Since the results obtained in this study suggested that TL-specific CTL clones recognized the framework of TL, we analyzed their CDR3 lengths to verify whether they resemble those of TCR{gamma}{delta} T cells/Ig or conventional TCR{alpha}ß T cells. As shown in Table 1, six TL-specific CTL clones were found to all use {alpha} and ß TCR polypeptides that have nearly identical CDR3 lengths, similar to conventional TCR{alpha}ß T cells. The CDR3 lengths of several TCR{alpha}ß CTL clones which recognize H-2Kb TAP-dependently or -independently (10) were also analyzed, and similar results were also obtained. These findings altogether show that TL-specific TCR{alpha}ß CTL recognize epitopes on the framework of the {alpha}1/{alpha}2 domain of TL without the antigenic molecules presented by these domains, but they seem to use {alpha} and ß TCR polypeptide chains with conventional CDR3 lengths. In addition, this study demonstrated the heterogeneity of V{alpha}Vß usage of these CTL clones.


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Table 1. CDR3 lengths of CTL clones
 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We previously reported that most TL-specific CTL recognize the epitope(s) expressed on TL in a TAP-independent manner (10). In the present study, using TL tetramers free from any antigenic molecules, which were prepared with recombinant T3b-TL and ß2-microglobulin derived from E. coli, we demonstrated that CD8-dependent TL-specific TCR{alpha}ß CTL recognize the framework of the TL molecules without antigenic peptides. In addition, the results suggested that post-translational modifications by mammalian and/or insect cells are not involved in the epitope formation. These results are consistent with the recent reports that TL has no antigen-binding groove wide enough for a peptidic or lipid antigen (14) and can demonstrate a good conformation for binding to CD8{alpha}{alpha} expressed on iIEL even without peptides (15).

Our present study on the reactivity of TL-specific CTL clones against various TL chimeric molecules suggested that they recognize the framework of the {alpha}1/{alpha}2 domain as an epitope. At present, we assume that the CTL recognize the conformational epitopes of TL, because neither acid-treated TL nor linear short polypeptides of TL (32) can stimulate TL-specific CTL (unpublished observations). With regard to the recognition mode, TL-specific TCR{alpha}ß CTL recognizing the framework of the TL molecule may be considered unique, resembling TCR{gamma}{delta} T cells and Ig rather than conventional TCR{alpha}ß T cells. On sequence analysis of TCR, however, all six CTL clones tested were shown to recognize an epitope of the {alpha}1/{alpha}2 domain of the TL molecule using {alpha} and ß TCR polypeptides whose CDR3 lengths were nearly identical to each other, like most TCR{alpha}ß T cells (25). The present TCR analysis also showed heterogeneity of V{alpha}Vß usage of these CTL clones (Table 1), in contrast to the NKT cell population expressing an invariant TCR chain like V{alpha}14 (V{alpha}24 in humans) for CD1d (33,34), suggesting the possibility that the CTL clones recognize multiple epitopes on the framework of the TL molecule, although we have no direct evidence to prove it.

The present study showed that substitution of the {alpha}3 domain of TL with that of HLA-A2 or -E (TL/HLA tetramers), but not H-2Kb, results in loss of the binding capacity to TL-specific CTL, suggesting that a mouse-specific sequence(s) of the {alpha}3 domain of TL may be involved in the epitope(s). If this were the case, the epitope(s) would be expected to consist of the TL-specific sequence of the {alpha}2 (and/or {alpha}1) domain of T3b-TL and the mouse specific sequence of {alpha}3 domain shared by both T3b-TL and H-2Kb, but not by HLA. However, this possibility is probably not so high, because even a minor population of TL-specific bulk CTL, speculated to recognize various epitopes on TL, seems not to be stained with chimeric TL/HLA tetramers (unpublished observations). A more likely explanation for the loss of the ability to activate TL-specific CTL by TL/HLA chimeras is that, even if the {alpha}1/{alpha}2 domain of the TL molecule may be recognized by TL-specific CTL clones, the binding affinity of the TCR of these CTL clones alone to the corresponding TL epitope is not sufficient for activation. Accordingly, only when binding of the CD8 molecule to the {alpha}3 domain of TL or Kb molecule occurs can the CTL clones be activated, i.e. in the case of the TL/A2 chimeric molecule, the {alpha}3 domain of HLA has very weak binding affinity to the murine CD8 (2628) so that it cannot help these CTL clones to express cytotoxic activity even when their TCR can recognize the corresponding TL epitopes. It should be noted in this regard that the capacity of binding of the {alpha}3 domain of H-2Kb to the mouse CD8 molecule is known to be weaker than that of TL (6), but much stronger than the case of HLA (2628), supporting the present finding that the {alpha}3 domain of H-2Kb is sufficient for the binding and activation of TL-specific CTL clones.

TL-specific CTL derived from C3H/He are considered to be selected in the thymus based on affinity for H-2Kk and/or Dk, since they do not express T3k-TL in the thymus. Therefore, the affinity of the TCR of these clones for TL is a very interesting issue. It has been reported that CD8-independent CTL clones in general have TCR of higher avidity for their ligands than CD8-dependent counterparts (3537). We previously reported that TL-specific CTL can also be divided into CD8-dependent and -independent groups based on the results of antibody-blocking tests (9), but the relation between the avidity and CD8 dependency of TL-specific CTL clones seems to be quite different from the previous reports on conventional MHC class Ia-restricted CTL clones (3537), i.e. CD8-dependent TL-specific CTL clones seem to have higher overall avidity than CD8-independent ones for TL, since the former are stained more brightly with TL tetramers and show stronger cytotoxic activity CTL (5,9). Our present results suggest that the affinity of the TCR of CD8-dependent TL-specific CTL clones is similar to that of CD8-dependent MHC class Ia-restricted CTL, and that they can be stained with TL tetramers only when CD8 stabilize the interaction between TCR and TL. In the case of CD8-independent TL-specific CTL clones, the binding affinity of TCR may be too low to initiate recruitment and/or rearrangement of CD8 for stable interaction with TCR (38), and other molecules may be used for their adhesion and/or activation. In other words, these CTL clones are CD8 independent, just because the TL–CD8 interaction may not be involved in their adhesion and/or activation. Supporting this idea, LFA-1–ICAM-1 interaction was found to be necessary for the execution of cytotoxic responses (mainly through the Fas–Fas ligand pathway) by CD8-independent TL-specific CTL clones (9).

Recent studies by ourselves and others demonstrated that TL tetramers can bind to iIEL and thymocytes in a CD8-dependent, but TCR/CD3-independent, manner, while binding to TL-specific CTL is TCR/CD3 and CD8 dependent (5,6). Here, we provide experimental evidence to suggest that TCR and CD8 bind predominantly to the {alpha}1/{alpha}2 and {alpha}3 domains of the TL respectively. Very recently, Leishman et al. clearly showed that TL tetramers bind to CD8{alpha}{alpha} expressed on iIEL by demonstrating stronger binding of TL to the CD8{alpha}{alpha} homodimer than the CD8{alpha}ß heterodimer with surface plasmon resonance (6). It is of interest whether the high-avidity interaction between TL and CD8{alpha}{alpha} may affect the positive and/or negative selection of iIEL recognizing TL with TCR. TL-reactive T cell lines recognizing TL with TCR have never been generated from iIEL populations. One possible explanation is that TL-reactive T cells (recognizing TL with TCR) may be eliminated in the gut, but not in the thymus. Such a differential negative selection might occur because the overall avidity of TCR plus CD8{alpha}{alpha} for TL in the gut is assumed to be higher than that of TCR plus CD8{alpha}ß in the thymus. If such a selection does occur, it would appear to be unique to the TL system, since the affinity of most MHC class I molecules for CD8{alpha}{alpha} and CD8{alpha}ß is comparable and lower than that of TL for CD8{alpha}{alpha} (16). Alternatively, TL-reactive T cells might exist in iIEL populations, but their cytotoxic activity could be suppressed through TL–CD8{alpha}{alpha} interaction (6). We are now attempting to generate transgenic mice expressing TL-specific TCR to address these intriguing issues.

In conclusion, the present study showed that TL-specific CTL recognize the {alpha}1/{alpha}2 domain of the L framework with the help of CD8 binding to the mouse-specific sequence of the {alpha}3 domain, common to TL and Kb, but not to HLA-A2 or -E.


    Acknowledgements
 
This work was supported in part by a Grand-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science, and a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology, Japan. We are grateful to Drs K. Itoh and E. Nakayama for their kind provision of various materials used in this study. We thank Drs A. Attinger, H. Cheroutre, and M. Kronenberg for their valuable discussions and suggestions and also Dr M. A. Moore for his editorial assistance.


    Abbreviations
 
6xHis—histidine tag

BSP—biotin-specific peptide

CTL—cytotoxic T lymphocyte

GS—GlySer linker

iIEL—intestinal intraepithelial lymphocyte

TL—thymus leukemia antigen


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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