Positive Selection of gamma delta CTL by TL Antigen Expressed in the Thymus

By Kunio Tsujimura,* Toshitada Takahashi,* Akimichi Morita,*Dagger Hitomi Hasegawa-Nishiwaki,* Shigeru Iwase,*§ and Yuichi Obata*

From the * Laboratory of Immunology, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya 464, Japan; Dagger  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

Summary
Materials and Methods
Results
Discussion
Footnotes
Acknowledgements
References


Summary

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 TCRgamma delta , whereas almost all those from C3H expressed TCRalpha beta . 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 gamma delta CTL recognizing TL in Tg.Tlaa-3-2, but not C3H mice, was confirmed by the establishment of CTL clones. A total of 14 gamma delta CTL clones were established from Tg.Tlaa-3-2, whereas none were obtained from C3H. Of the 14 gamma delta 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 TCRgamma delta and CD3, and also by antibodies to CD8alpha and CD8beta in CD8+ clones, showing that the activity was mediated by TCRgamma delta and coreceptors. The thymic origin of these gamma delta CTL clones was indicated by the expression of Thy-1 and Ly-1 (CD5), and also CD8alpha beta heterodimers in CD8+ clones on their surfaces and by the usage of TCR Vgamma 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 gamma delta 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 alpha beta or gamma delta 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 gamma delta T cells has been suggested by the distinctive tissue distribution and limited V gene usage of gamma delta T cells. Indeed, several studies have shown that gamma delta T cells recognize nonclassical class I molecules encoded by genes located within the Q or T regions (reviewed in references 4, 5).

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 TCRalpha beta 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.

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)-Lyt2(CD8)- double negative (DN) cells of gamma delta 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 gamma delta 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.


Materials and Methods

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.


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.
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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 TCRalpha beta (H57-597; Dr. R.T. Kubo, National Jewish Center for Immunology and Respiratory Medicine, Denver, CO [18]), hamster mAb to TCRdelta (3A10; Dr. S. Tonegawa, Massachusetts Institute of Technology, Cambridge, MA [19]), hamster mAb to TCRgamma delta (GL3; Cedarlane Labs. Ltd., Hornby, Ontario, Canada), hamster mAb to Vgamma 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).

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 × (- 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.

For cloning, cells were diluted to 1-100 cells/well and cultured with irradiated Tg.Con.3-1 spleen cells in the presence of IL-2 in 96-well plates. After 10-14 d, the cytotoxity of clonally growing cells was tested. Established clones were maintained by weekly stimulation and their activity was tested as described above.

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). TCRgamma chains were amplified using Vgamma -specific primers and a single common Cgamma primer by the methods of Takagaki (21). The nomenclature of Vgamma is according to Reilly et al. (22). The RT-PCR products were separated by agarose gel electrophoresis.

Nucleotide Sequencing of the Vgamma -Jgamma 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).


Results

Characteristics of Anti-T3b-TL CTL Generated in Transgenic Mice Expressing Tlaa-3-TL Antigen in the Thymus.

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 TCRalpha beta , while ~15% from Tg.Tlaa-3-2 and >20% from Tg.Tlaa-3-1 expressed TCRgamma delta . In the gamma delta 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 gamma delta 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 alpha beta 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 alpha beta 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).


Fig. 2. Experimental design for testing the characteristics of anti-TL CTL generated in Tlaa-3 transgenic and C3H mice. The skins obtained from Tg.Con.3-1 mice expressing T3b-TL antigen were grafted onto Tg.Tlaa-3-1 and Tg.Tlaa-3-2 transgenic and C3H mice. Tg.Tlaa-3-1 and Tg.Tlaa-3-2 mice express Tlaa3-TL encoded by the transgene in the thymus and in the intestines as well as endogenous T3kTL in the intestines, while C3H mice express T3k-TL in the intestines, but not in the thymus. CTL were generated by MLC, in which spleen cells of mice that had rejected the skin graft were restimulated with irradiated Tg.Con.3-1 spleen cells.
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Fig. 3. Surface antigen profiles of the TL-reactive cells obtained from bulk MLC. Spleen cells from Tg.Tlaa-3-2, Tg.Tlaa-3-1, and C3H that had rejected Tg.Con.3-1 skin grafts were cultured with irradiated spleen cells from Tg.Con.3-1. After 5 d in culture, the cells were double-stained with various combinations of antibodies, and the blastic cell population was gated and analyzed with a FACScan®. (A) Expression of TCR/CD3 complexes on Thy-1+ cells in bulk MLC. About 80, 25, and 80% of the gated cells derived from Tg.Tlaa-3-2, Tg.Tlaa-3-1, and C3H, respectively, expressed Thy-1 on their surfaces. The percentages of cells expressing CD3, TCRalpha beta , and TCRgamma delta among Thy-1+ cells are indicated. (B) Expression of L3T4 and Lyt-2 on TL-reactive gamma delta T cells obtained from Tg.Tlaa-3-2.
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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-TCRalpha beta , CD3, and Lyt-2 antibodies but not by anti-TCRgamma delta or anti-L3T4. In the Tg.Tlaa-3-2 case, the CTL activity was blocked partially by anti-TCRgamma delta antibody, although not as strongly as shown by anti-TCRalpha beta antibody, indicating a certain population of gamma delta 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 gamma delta lineage by the presence of Tlaa-3-TL antigen in the thymus.


Fig. 4. Specificity of the TL-reactive CTL populations in bulk cultures derived from Tg.Tlaa-3-2 and C3H. (A) Generation of CTL against TL antigen in Tg.Tlaa-3-2 and C3H. The effector cells were prepared from Tg.Tlaa-3-2 and C3H mice that had rejected Tg.Con.3-1 skin grafts. Target cells were T3b-TL+ blast cells of Tg.Con.3-1 (bullet ) and TL- blast cells of C3H (open circle ), H-2Kb/T3b Ltk- transfectants (black-square), Ltk- (square ), ERLD (black-triangle), RADA1 (triangle ), and ASL1 (down-triangle). For TL expression on these target cells, see Materials and Methods. (B) Antibody blocking for characterization of the CTL populations from Tg.Tlaa-3-2 and C3H. The cytotoxic activity of CTL generated in Tg.Tlaa-3-2 and C3H by grafting Tg.Con.3-1 skin was tested in the presence of mAbs to TL.2 (TT213) (square ), TCRalpha beta (open circle ), TCRgamma delta (bullet ), CD3 (ccirf ), L3T4 (black-down-triangle ), Lyt-2 (black-triangle), or cAb to H-2k (black-square). The broken lines indicate the percentage specific lysis obtained without antibodies. The target cells were Con A blast cells of Tg.Con.3-1 and E/T ratio was 15:1.
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Establishment of gamma delta CTL Clones.

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 TCRgamma delta , whereas all C3H CTL clones expressed TCRalpha beta . Thus, establishment of the CTL clones indicated that gamma delta CTL are induced characteristically in mice expressing TL in their thymus, confirming the results of bulk cultures.

Characteristics of the gamma delta CTL Clones.

The three gamma delta CTL clones described above were all Lyt-2+ (see below), although one third of gamma delta T cells in bulk culture were DN as shown in Fig. 3 B. To test whether TL-reactive gamma delta CTL clones with DN phenotype could be established, 11 additional gamma delta CTL clones were derived from five Tg.Tlaa-3-2 mice. Thus, a total of 14 gamma delta 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 CD8alpha chain, and in T cells of thymic origin, it forms a heterodimer with Lyt-3, a CD8beta chain, while it forms an alpha alpha homodimer in those of intestinal epithelium (23). All Lyt-2+ gamma delta 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 gamma delta 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 gamma delta T cells of thymic origin, but not on those of extra-thymic origin (24, 25).

Table 1. Summary of gamma delta CTL Clones


Clone* Vgamma usage (RT-PCR)  gamma delta TCR expression§ Surface antigens (FACS®) Blocking of the cytotoxicity with antibody
TCRgamma delta TCRdelta Vgamma 4 CD4/CD8par Thy-1 Ly-1 TL H-2 TCRdelta gsim gsim Lyt-2 Lyt-3 L3T4

KC1-3     1/2Dagger + +  - CD8alpha beta + + +  - + + +  -
  1-7     4Dagger + + + CD8alpha beta + + +  - + + +  -
  4-4     2/4Dagger + + + CD8alpha beta + + +  - + + +  -
  4-8 1/2/4Dagger + + + DN + + +  - +  -  -  -
  4-9   2/4 + + + CD8alpha beta + + +  - + + +  -
  5-1   2/4 + + + CD8alpha beta + + +  - + + +  -
  5-2   2/4 + + + CD8alpha beta + + +  - + + +  -
  6-1   2/4Dagger + + + DN + + +  - +  -  -  -
  6-2   2/4 + + + CD8alpha beta + + +  - + + +  -
  7-3   1/2/4 + + + CD8alpha beta + + +  - + + +  -
  7-4   1/2Dagger + +  - DN + + +  - +  -  -  -
  8-1   2/4 + + + DN + + +  - +  -  -  -
  8-3   2/4Dagger + + + DN + + +  - +  -  -  -
  8-4   2/4 + + + DN + + +  - +  -  -  -

*  The first digit of the clone name shows the origin of the mouse.
Dagger  RT-PCR products were sequenced (see Fig. 6).
§  Expression of TCRgamma delta , TCRdelta , and Vgamma 4 chains on the cell surface were detected by FACS® using moAb GL3, 3A10, and UC3-10A6, respectively.
par  Monoclonal antibodies to L3T4, Lyt-2 and Lyt-3 were used.
 The target cells were Con A blasts of spleen cells from Tg.Con.3-1 (T3b-TL positive).
**  Monoclonal antibody 3A10 was used.


Fig. 6. Nucleotide sequences of the V-J junction of TCRgamma -genes expressed in gamma delta CTL clones. RNA was extracted from gamma delta CTL clones and RT-PCR was carried out. Sequencing of the RT-PCR products was conducted as described in Materials and Methods. For each sequence, the N nucleotides were deduced from the germline sequence previously reported (26). The nomenclature of Vgamma segments is according to Reilly et al. (22).
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The 14 gamma delta 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 gamma delta 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+ gamma delta CTL clones, KC4-4, and Fig. 5, B and D, for one of the DN gamma delta CTL clones, KC8-3. The CTL activity of all these gamma delta 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 gamma delta CTL clones directly recognize the TL molecule. The CTL activity was also blocked by anti-TCRgamma delta and CD3 antibodies. All Lyt-2+ gamma delta 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 gamma delta CTL clones showed no differences in their expression of adhesion, costimulatory, and accessory molecules; all gamma delta CTL clones were positive for Pgp-1 (CD44), LFA-1 (CD11a), ICAM-1 (CD54), CD2, CD28, CTLA-4, and IL-2Ralpha /beta , and negative for L-selectin (CD62L), VLA-4alpha (CD49d), and HSA (data not shown).


Fig. 5. Specificity of two representatives of Lyt-2+ and DN gamma delta CTL clones. (A and B) Target specificity of Lyt-2+ KC4-4, and DN KC8-3 gamma delta CTL clones against TL. Target cells were Con A blast cells of Tg.Con.3-1 (bullet ), C3H (open circle ) and B6 (ccirf ), H-2Kb/T3b Ltk- transfectants (black-square), Ltk- (square ), and RADA1 (triangle ). For TL expression on these target cells, see Materials and Methods. (C and D) Antibody blocking for characterization of Lyt-2+ KC4-4 and DN KC8-3 gamma delta CTL clones. The cytotoxic activity of gamma delta CTL clones was tested in the presence of mAb to TL.2 (TT213)(square ), TCRalpha beta (open circle ), TCRgamma delta (bullet ) , CD3 (ccirf ), L3T4 (black-down-triangle ), Lyt-2 (black-triangle), Lyt-3 (triangle ), or cAb to H-2k (black-square). The target cells were Con A blast cells of Tg.Con.3-1 and E/T was 5:1.
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To further confirm the thymic origin of these gamma delta CTL clones, their Vgamma segment usage was determined. By RTPCR, nine clones yielded Vgamma 2 and Vgamma 4 products, two clones Vgamma 1 and Vgamma 2, two clones Vgamma 1, Vgamma 2 and Vgamma 4, and one clone Vgamma 4, while no clones yielded Vgamma 5, Vgamma 6, or Vgamma 7 products (Table 1). There was no distinctive difference in the usage of Vgamma by Lyt-2+ clones from that by DN clones. All of 12 clones with RT-PCR Vgamma 4 products reacted with an antibody specific to Vgamma 4 in the flow cytometric analysis, indicating that Vgamma 4 chains were indeed expressed on the cell surface. It has been reported that the Vgamma 4 chain is preferentially used by thymus-dependent gamma delta T cells, while Vgamma 1 and Vgamma 2 chains are used by both thymus-dependent and -independent gamma delta 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 gamma  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 Vgamma 2 and Vgamma 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 Vgamma 2 chains in the KC6-1 and KC7-4 clones, suggesting that a diverse set of gamma delta TCR recognize the TL molecule. In summary, the usage of Vgamma 4 chains with N sequences in 12 of 14 clones, and the expression of Thy-1, Ly-1, and CD8alpha beta heterodimer in the case of Lyt-2+ clones, indicated that these gamma delta CTL clones that recognize TL were matured in adult thymus.


Discussion

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 gamma delta T cells, in contrast to those in C3H mice which were found to be mostly alpha beta T cells. Most of the gamma delta CTL were of adult thymic origin; all of them expressed Thy-1, Ly-1 (CD5) and CD8alpha beta heterodimers in the cases of Lyt-2+ clones, and 12 of 14 clones used TCR Vgamma 4 chains with N sequences in the V-J junctions except for Vgamma 4 of KC4-8. Thus, our results indicate that the presence of Tlaa-3-TL in the thymus promotes the positive selection of gamma delta 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 gamma delta T cells, selected on the basis of their affinity for Tlaa-3, are able to react with T3b as well, and that such gamma delta 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 gamma delta 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 gamma delta 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 gamma delta T cells has also been speculated from the observation that it is expressed in the intestinal epithelium and gamma delta T cells are present nearby (9, 10), although no direct evidence for any interaction between the intestinal epithelium and gamma delta T cells has been presented.

It is an intriguing question whether normal mice that express TL in the thymus also produce gamma delta 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 gamma delta 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 gamma delta 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 gamma delta T cells in mice and humans and most gamma delta T cell clones or hybridomas reported so far do not express either CD4 or CD8 (4, 5). This suggests that gamma delta 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 gamma delta T cells do not require the presence of CD4 or CD8 molecules. Since there have hitherto been only few reports of gamma delta T cell clones with clear CTL or helper activity, it has been difficult to conduct detailed studies on the requirement of coreceptors for gamma delta T cells. Our present finding that 8 of 14 TL-restricted gamma delta CTL clones express CD8alpha beta , supports the proposal that TL can bind CD8 (34) and further implies that the CD8-p56lck signal pathway can operate in gamma delta CTL, similarly to the alpha beta CTL case (35). In addition to CD8+ gamma delta CTL, DN gamma delta CTL clones were also established. Analysis of DN gamma delta CTL clones has so far shown no differences from CD8+ gamma delta 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 gamma delta CTL may be due to the affinity of TCR for the target, or to the density of antigenic epitopes. Measurement of the affinity of TCRgamma delta expressed in CD8+ and DN CTL clones, and detailed analysis of the target molecules of these gamma delta CTL, are now needed to clarify these issues.

Most of our sequenced gamma delta CTL clones were found to have two in-frame TCR gamma  chain transcripts. Further studies with transgenic mice are needed to determine which inframe gamma  chain is responsible for TL recognition, although surface expression of the Vgamma 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 gamma  chains, although analysis of delta  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 gamma delta and alpha beta 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 TCRalpha beta or TCRgamma delta . 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 alpha 1 and alpha 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.


Footnotes

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.
   1Abbreviations used in this paper: B6, C57BL/6; C3H, C3H/He; cAb, conventional antibody; DN, CD4-CD8- double negative; Ltk-, thymidine kinase-negative L cell; MFI, mean fluorescence intensity; PBR, peptide binding region; RT, reverse transcription.

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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