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Identification of a New Type of Invariant V14⁺ T Cells and Responsiveness to a Superantigen, Yersinia pseudotuberculosis- Derived Mitogen¹

Junji Yagi^2,*, Umberto Dianzani^§, Hidehito Kato^*, Toshihiro Okamoto, Tomoko Katsurada^*, Donatella Buonfiglio^§, Tohru Miyoshi-Akiyama^* and Takehiko Uchiyama^*,

Departments of ^* Microbiology and Immunology and Oral and Maxillofacial Surgery, and Institute of Laboratory Animals, Tokyo Women’s Medical University, Tokyo, Japan; and ^§ Laboratory of Immunology, Department of Medical Science, A. Avogadro University of Eastern Piedmont, Novara, Italy

	Abstract

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We examined the expression of the H4 T cell activation marker in thymic T cell subpopulations and found that TCR-ß⁺ CD4⁺ thymic T cells are segregated into three subpopulations based upon H4 levels. Thymic T cells with either no or low H4 expression differentiate via the mainstream differentiation pathway in the thymus. H4^int thymic T cells, which express a skewed Vß repertoire of Vß2, -7, and -8 in their TCRs, show the phenotype of NKT cells: CD44^high, Ly6C^high, NK1.1⁺, and TCR-ß^low. H4^high thymic T cells also show a skewed Vß repertoire, Vß2, -7, and -8, and predominantly express an invariant V14-J281⁺ -chain in their TCRs but constitute a distinct population in that they are CD44^int, Ly6C^-, NK1.1^-, and TCR-ß^high. Thus, invariant V14⁺ thymic T cells consist of ordinary NKT cells and a new type of T cell population. Vß7⁺ and Vß8.1⁺ invariant V14⁺ thymic T cells are present in DBA/2 mice, which carry mammary tumor virus-7-encoded superantigens, in comparable levels to those in BALB/c mice. Furthermore, Vß7⁺ invariant V14⁺ thymic T cells in DBA/2 mice are in the immunologically responsive state, and Yersinia pseudotuberculosis-derived mitogen-induced Vß7⁺ invariant V14⁺ thymic T cell blasts from DBA/2 and BALB/c mice exhibited equally enhanced responses upon restimulation with Y. pseudotuberculosis-derived mitogen. Thus, invariant V14⁺ thymic T cells that escape negative selection in DBA/2 mice contain T cells as functionally mature as those in BALB/c mice.

	Introduction

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CD4 single-positive (SP)³ and CD8 SP T cells differentiate from CD4⁺ CD8⁺ double-positive (DP) thymocytes via the CD4/CD8 pathway in the thymus (1, 2). Two selective events, elimination of self-reactive T cells (negative selection) and survival of T cells that can interact with foreign peptide/MHC complex (positive selection), occur at the stage of DP thymocytes (3, 4, 5). Negative selection is well documented in mice expressing endogenous superantigens that are encoded by mouse mammary tumor viruses (Mtvs) and that eliminate developing T cells in a Vß-specific fashion (6, 7). Mtv-7-encoded superantigens, for instance, delete Vß6⁺, Vß7⁺, Vß8.1⁺, and Vß9⁺ T cells (8). Endogenous superantigens do not always delete all Vß-specific thymocytes. Blackman and coworkers have previously reported that Mtv-7⁺ TCR ß-chain transgenic mice show only partial deletion of thymocytes expressing the relevant TCR Vß (9, 10), and that TCR V usage in T cells surviving the negative selection is biased in those mice (11). Thus, although TCR Vß element plays the dominant role in T cell response to superantigens, accumulating evidence has shown that non-Vß TCR element such as -chain contributes to superantigen responsiveness (12, 13, 14) and influences the susceptibility to negative selection (10, 11, 12, 15). In addition to conventional T cells, a specialized population of TCR-ß⁺ T cells that presumably do not differentiate via the CD4/CD8 pathway resides in the thymus as a small population (1%). They express NK surface receptors, and are hence called NKT cells, and a highly restricted TCR composed of an invariant -chain (V14-J281 in mice) and a ß-chain with restricted Vß (Vß2, -7, and -8 in mice) and heterogeneous Dß and Jß elements (16, 17).

Several studies have shown that Vß8.1⁺ NKT cells are present in the thymus of Mtv-7⁺ mice at an equal level to Mtv-7^- mice (18, 19). Thus, Vß8.1⁺ NKT cells escape the negative selection by "self-Ags," Mtv-7-encoded superantigens. However, it is not currently elucidated whether invariant TCR -chain in NKT cells contributes to the escape of Vß8.1⁺ NKT cells from the negative selection. Further questions arise as to whether Vß7⁺ NKT cells also survive in Mtv-7⁺ thymus and whether Vß7⁺ and Vß8.1⁺ NKT cells in Mtv-7⁺ mouse are functionally mature, and how they are regulated to not respond harmfully to Mtv-7-encoded superantigens in vivo. Other contributors reported a T cell activation marker, H4, which comodulates and cocaps with TCR, indicating that the molecule physically associates with TCR (20). We became interested in whether the H4 molecule is expressed on NKT cells.

In the present study, we examined the expression of H4 molecule in thymic T cell subpopulations and found that thymic T cells with an intermediate amount of H4 expression are ordinary NKT cells, whereas thymic T cells with a high amount of H4 expression constitute a new type of invariant V14⁺ T cell. Vß7⁺ and Vß8.1⁺ invariant V14⁺ thymic T cells were present in Mtv-7⁺ DBA/2 mice at similar levels to Mtv-7^- BALB/c mice. Furthermore, Vß7⁺ invariant V14⁺ thymic T cells in DBA/2 mice showed substantial and comparable responses with those in BALB/c mice upon stimulation with a superantigen, Yersinia pseudotuberculosis-derived mitogen (YPM) (21, 22, 23). Based upon these results, we discuss the immune regulation of the potentially self-reactive T cells.

	Materials and Methods

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Animals

BALB/c and C57BL/6 mice were bred in our own colony at the Department of Microbiology and Immunology, Tokyo Women’s Medical University. DBA/2 mice were purchased from Japan S.L.C. (Hamamatsu, Japan). Female mice, 6–7 wk old, were used in this study.

YPM and Abs

The YPM superantigen was purified from extract of Escherichia coli XL1-Blue carrying pQE30-6xH·ypm (22) using Ni-NTA Agarose (Qiagen, Chatsworth, CA) followed by Sepharose Fast Flow (Pharmacia LKB Biotechnology, Tokyo, Japan). YPM stimulates murine Vß7- and Vß8-bearing T cells (23). The C398.4A mAb specific for H4 was produced in Armenian hamsters by immunizing with the murine T cell clone D10.G4.1 as described previously (20). mAbs to CD8 (83.12.5 and 53-6.7), Vß2 (B20.6), Vß4 (KT4-10), Vß6 (RR4-7), Vß7 (TR310), Vß8.1+8.2+8.3 (F23.1), Vß8.1+8.2 (KJ16), Vß8.2 (F23.2), Vß9 (MR10-2), Vß10 (KT10b-2), Vß14 (14.2), and Thy1.2 (HO13) were described previously (23, 24, 25). mAbs to TCR-ß (H57-597) and CD44 (IM7), PE-anti-CD4 (RM4-4), cy-chrom-streptavidin and biotinylated anti-Ly6C (AL-21), and anti-NK1.1 (PK136) were purchased from PharMingen (San Diego, CA). FITC-anti-CD8 (53-6.7) and PE-streptavidin were obtained from Becton Dickinson (Mountain View, CA). The following anti-IgG Abs were used: FITC-goat anti-rat IgG from Zymed Laboratories (South San Francisco, CA), FITC-goat anti-mouse IgG from Biosource (Camarillo, CA), FITC-goat anti-hamster IgG from Southern Biotechnology Associates (Birmingham, AL), and goat anti-rat IgG from Organon Teknika (Westchester, PA). Purified hamster IgG and anti-H4 mAb were conjugated with biotin in our laboratory.

Culture medium

RPMI 1640 supplemented with 100 µg of streptomycin per ml, 100 U of penicillin per ml, 10% fetal bovine serum, and 5 x 10^-5 M 2-ME was used for the culture.

Flow cytometry analysis

To detect expression of H4 in thymic T cell subpopulations, thymocytes were stained by incubation with biotinylated anti-H4 mAb followed by a cocktail of FITC-anti-CD8, PE-anti-CD4, and cy-chrom-streptavidin. The relationship between the expression of H4 and various surface molecules was analyzed as follows. Thymocytes were stained with a combination of unconjugated Abs or anti-H4 mAb and appropriate FITC-anti-IgG and were incubated with an excess amount of normal hamster IgGs to block unbound binding sites on FITC-anti-IgG. After washing, FITC-stained cells were incubated with a combination of biotinylated anti-H4 mAb or biotinylated Abs and PE-streptavidin. To detect expression of H4 in CD4^-CD8^- double-negative (DN) thymocytes, FITC-stained cells with anti-Vß Abs and appropriate FITC-anti-IgG were incubated with biotinylated anti-H4 mAb followed by a combination of PE-anti-CD4 and cy-chrom-streptavidin. The purity of cells after the preparation was checked by incubation of cells with unconjugated anti-Vß Abs or anti-H4 mAb followed by appropriate FITC-anti-IgG, or a combination of FITC-anti-CD8 and PE-anti-CD4. Samples of number of viable cells indicated were analyzed by an Epics CS flow cytometer (Coulter Immunology, Hialeah, FL), Epics XL flow cytometer (Coulter Immunology), or FACScan (Becton Dickinson, Cockeysville, MD).

Treatment of mice with dexamethasone

Mice were treated with dexamethasone (Sigma, St. Louis, MO) as reported by Screpanti et al. (26). Various doses indicated in 40% ethanol/saline were injected s.c. Thymuses were taken 2 days later for the preparation of cells.

Preparation of cells

Single thymocyte suspensions were prepared in Hanks’ solution (Nissui Pharmaceutical, Tokyo, Japan) with 2% FCS. CD4 SP+DN thymocytes were obtained from BALB/c and C57BL/6 mice by two rounds of treatment of thymocytes with 83.12.5 and guinea pig complement (C') (CD8⁺ cells, <0.2%). DBA/2 CD4 SP+DN thymocytes were obtained by two rounds of indirect immunomagnetic depletion according to the manufacturer’s instructions. Briefly, after thymocytes were incubated with 53-6.7, 53-6.7-coated cells were rosetted with sheep anti-rat IgG bound magnetic beads (Dynabeads, Dynal, Great Neck, NY) and separated with a magnet (CD8⁺ cells, <0.1%).

To obtain Vß7⁺ invariant V14⁺ thymic T-enriched population, BALB/c and DBA/2 CD4 SP+DN thymocytes were obtained by one round of treatment with 83.12.5 and C' and by taking nonadherent 53-6.7-treated cells from goat anti-rat IgG (30 µg/ml)-coated dishes (no. 25020; Corning, Corning, NY), respectively. Vß7⁺ thymic T cells were then enriched by treatment with a cocktail of anti-Vß mAbs (anti-Vß4, -8, -10, and -14) and sheep anti-rat IgG-beads and sheep anti-mouse IgG-beads (Dynabeads, Dynal). These cells were further incubated with anti-H4 mAb followed by anti-mouse IgG-beads, and H4⁺ cells were positively selected with a magnet. After overnight culture, cells free from beads were separated with a magnet.

To obtain CD4⁺ Vß7⁺ mainstream thymic T-enriched population, CD4 SP+DN thymocytes obtained from 500 µg of dexamethasone-treated BALB/c mice were incubated with a cocktail of anti-Vß mAbs and anti-H4 mAb, and Ab-coated cells were depleted using anti-IgG-beads as described above. T-depleted spleen cells were obtained by treatment of spleen cells with anti-Thy1.2 and C' followed by inactivation with mitomycin C (Kyowa Hakko Kogyo, Tokyo, Japan) and used as APCs.

RT-PCR

Total mRNA was extracted by oligo(dT)-latex (Nippon Roche, Tokyo, Japan) from thymic T cell blasts and reverse transcribed into cDNA at 42°C for 2 h using RAV-2 reverse transcriptase (Takara Biomedicals, Osaka, Japan) and random hexamer primers (Takara Biomedicals). The cDNA was amplified on a thermocycler (programmable temp-control system PC-700, Sci-media, Tokyo, Japan) using Taq DNA polymerase (Takara Biomedicals) and oligonucleotides pairs specific for Vß7 and Cß or those for a panel of V and C primers (Vß7, 5'-TACAGGGTCTCACGGAAGAAGCG-3'; Cß, 5'-CTGCTCGGCCCCAGGCCTCT-3'; V2, 5'-AGCACTTTTAACTACTTCCCA-3'; V8, 5'-AATATCTCAACGAAGCCCCT-3'; V10, 5'-CGCAGCTCTTTGCACATTTC-3'; V11, 5'-GTTCTGCTCTGAGATGCAAT-3'; V14, 5'-AGTGTGACCCCCGACAAC-3'; C, 5'-TTGCTCTTGGAATCCATAGCT-3'). PCR amplification using Vß7 and Cß primers was performed for 28 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min, followed by a final extension of 72°C for 10 min. The PCR condition using primers of a panel of V and C primers was 30 cycles of 94°C for 30 s, 62°C for 30 s, and 72°C for 1 min. PCR products were separated by electrophoresis on a 2.0% agarose gel and visualized by UV light after ethidium bromide staining.

Cloning and sequencing of TCR junctional regions

DBA/2 and BALB/c CD4 SP+DN thymocytes were sorted into Vß7⁺ H4^high cells and Vß7⁺ H4^low cells, respectively, by an Epics CS flow cytometer. The sorted cells were stimulated with 3 µg/ml of YPM and syngenic APCs. After 3 days of culture, YPM-induced thymic T cell blasts were collected by applying the cell suspension to a Percoll gradient (density, 1.075) and expanded with 100 U/ml of human rIL-2 (a gift from Shionogi, Osaka, Japan) for an additional 2 days. First-strand cDNAs synthesized from mRNA from YPM-induced T cell blasts from Vß7⁺ H4^high thymocytes were amplified by PCR using V14 and C primers. The products were then ligated into pCR vector (Invitrogen, Carlsbad, CA) and were transfected into E. coli BL21 competent cells (Pharmacia Biotech, Uppsala, Sweden). After random selection of transformants, cloned plasmic DNAs were purified using Qiagen Plasmid Mini Kit (Qiagen). The products from the subsequent 25 cycles of PCR performed using cloned plasmic DNAs, V14 primers, and Dye Terminator Cycle Sequencing FC Ready Reaction Kit (PE Applied Biosystems, Foster City, CA) were sequenced by ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems). According to the procedure described above, junctional sequences of Vß7⁺ ß-chain in Vß7⁺ H4^high and Vß7⁺ H4^low cells were obtained using Vß7 and Cß primers.

Assay for the responsiveness of thymic T cell subpopulations

An indicated number of thymic T cell subpopulations were cultured with titrated amounts of YPM in the presence of 2 x 10⁵ syngenic APCs in flat-bottom microtiter plate (3072 Falcon, Becton Dickinson Labware, Oxnard, CA) in culture medium. After 2.5 days of culture, culture medium was removed for the determination of lymphokine concentration. In parallel with these assays, an indicated number of thymic T cells was cultured with 3 µg/ml of YPM in the presence of 5 x 10⁵ syngenic APCs in 48-well culture plate (3078 Falcon, Becton Dickinson Labware). After 3 days of culture, YPM-induced thymic T cell blasts were collected as described above and expanded with 100 U/ml of human rIL-2 for 4 more days. A total of 1 x 10⁵ YPM-induced thymic T cell blasts were restimulated with YPM as described above. After 24 h of culture, culture supernatants were obtained for the determination of lymphokine concentration.

Determination of lymphokine concentrations

Concentration of IL-2 in culture supernatants was determined in a bioassay as proliferation of IL-2-dependent CTLL-2 cells as described previously (21). IL-4 in culture supernatants was quantitated by sandwich ELISA according to the manufacturer’s instructions (PharMingen). Anti-mouse IL-4 (11B11) used as coating mAbs and biotinylated anti-mouse IL-4 (BVD6-24G2) used as detecting mAbs were purchased from PharMingen. Standard curves were generated using mouse rIL-4 (PharMingen).

Statistical analysis

Statistical significance between any two groups was analyzed by Student’s t test.

	Results

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Expression of H4 molecule in thymic T cell subpopulations

BALB/c thymic T cell subpopulations defined by CD4/CD8 phenotypes were analyzed for the expression of H4 molecule (Fig. 1, A and B). H4 is expressed in 25% of CD4 SP, 55% of CD8 SP, and 10% of DN thymocytes, but not at all in DP thymocytes. Based upon the amount of H4 expressed, CD4 SP thymocytes could be segregated into three groups; none to low, intermediate, and high expression, suggesting the heterogeneity of CD4 SP thymocytes. In CD8 SP thymocytes, only low H4 expression was observed. DN thymocyte expression of H4 ranged from low to high.

View larger version (24K): [in this window] [in a new window]   FIGURE 1. H4 expression in thymic T cell subpopulations. A, BALB/c thymocytes were stained with PE-anti-CD4, FITC-anti-CD8, and biotinylated anti-H4 mAb followed by cy-chrom-streptavidin. A sample of 300,000 viable cells was analyzed by FACScan. The histograms in B represent H4 expression (solid line) on CD4/CD8-defined subpopulations gated as indicated in A with background staining with biotinylated hamster IgG (dashed line). C, BALB/c CD4 SP+DN thymocytes were obtained and stained with a combination of anti-TCR-ß antibody/FITC-anti-hamster IgG and biotinylated anti-H4 mAb/avidin-PE. Samples of 50,000 viable cells were analyzed by an Epics CS flow cytometer. Circles in the figure show H4low, H4int, and H4high thymic T cells defined with differential expression of H4 on TCR-ß+ thymic T cells. Numbers indicate the percentages of these thymic T cell subpopulations in TCR-ß+ CD4 SP+DN thymocytes that were calculated by subtracting background staining. Data show typical results from one of four experiments.

Preferential expression of Vß2⁺, Vß7⁺, and Vß8⁺ TCR in H4^int and H4^high thymic T cells

We next performed TCR analysis of these thymic T cell subpopulations because H4^int thymic T cells express a low level of TCRs, similar to NKT cells (16, 19). The NKT cell population expresses a skewed Vß repertoire of Vß2, -7, and -8 in their TCRs (16, 17, 18, 19). Therefore, CD4 SP+DN thymocytes obtained from BALB/c thymocytes were stained with combinations of available anti-Vßs and anti-H4 mAb. The profiles in Fig. 2A indicate that H4^int and H4^high thymic T cells exclusively express Vß2⁺, Vß7⁺, and Vß8⁺ TCR, whereas H4^low thymic T cells express all of the TCR Vßs examined. The proportions of Vß7⁺, Vß8.2⁺, and Vß8.3⁺ cells in H4^high thymic T cells are 2- to 3-fold higher than those in whole TCR-ß⁺ thymocytes as controls, and those of Vß2⁺ and Vß8.1⁺ cells in H4^high thymic T cells are almost same as controls (Table I). The proportions of Vß2⁺, Vß7⁺, Vß8.1⁺, Vß8.2⁺, and Vß8.3⁺ cells in H4^int thymic T cells were equivalent to those in H4^high thymic T cells (data not shown). Thus, a biased usage of Vß2⁺, -7⁺, and -8⁺ TCR in H4^int and H4^high thymic T cells strongly suggests that NKT cells are enriched in these subpopulations. Furthermore, H4^int and H4^high thymic T cells in DN thymocytes expressed a skewed Vß repertoire of Vß7 and Vß8 TCR in their TCRs (Fig. 3), suggesting that these cells are also NKT cells. In C57BL/6 mice, H4^high thymic T cells are seen at much lower levels (0.7% in TCR-ß⁺ CD4 SP+DN thymocytes) (see the profiles in Fig. 4), and they show a biased usage of TCR Vß as in BALB/c mice (Table I).

View larger version (56K): [in this window] [in a new window]   FIGURE 2. Preferential expression of Vß2+, Vß7+, and Vß8+ TCR in H4int and H4high thymic T cells. BALB/c CD4 SP+DN thymocytes (A) or DBA/2 CD4 SP+DN thymocytes (B) were obtained and stained with combinations of anti-Vß or anti-TCR-ß/appropriate FITC-anti-IgG and biotinylated anti-H4 mAb/avidin-PE. Samples of 100,000 viable cells were analyzed by an Epics CS flow cytometer. In B, profiles of staining for representative Vß and H4 are shown. Data show typical results from one of three experiments.

View larger version (35K): [in this window] [in a new window]   FIGURE 3. Biased usage of Vß7+ and Vß8+ TCR in H4int and H4high thymic T cells in DN thymocytes. BALB/c CD4 SP+DN thymocytes were obtained and stained with anti-Vß/appropriate FITC-anti-IgG followed by biotinylated anti-H4 mAb/cy-chrom-streptavidin and PE-anti-CD4. Samples of 380,000 viable cells were analyzed by an Epics XL flow cytometer. The profiles in B represent Vß and H4 expression on DN thymocytes gated as indicated in A. In C, percentages of TCR Vß+ cells among H4high thymic T cells are shown. Data show typical results from one of three experiments.

View larger version (40K): [in this window] [in a new window]   FIGURE 4. Relationship between H4 and CD44, Ly6C, and NK1.1 expression. BALB/c (A) and C57BL/6 (B and C) CD4 SP+DN thymocytes were obtained and stained with combinations of anti-CD44/FITC-anti-rat IgG and biotinylated anti-H4 mAb/avidin-PE (A), and anti-H4 mAb/FITC-anti-hamster IgG and biotinylated anti-Ly6C/avidin-PE (B) or biotinylated anti-NK1.1/avidin-PE (C). Samples of 50,000 viable cells were analyzed by an Epics CS flow cytometer. Data show typical results from one of three experiments.

Relationship between H4 and other molecules associated with NKT cells

NKT cells have been defined with CD44^high, Ly6C^high, and NK1.1⁺ (16, 18, 19). To determine the expression of these molecules on H4^int and H4^high thymic T cells, CD4 SP+DN thymocytes from BALB/c and C57BL/6 mice were stained with combinations of anti-H4 mAb and anti-CD44, or anti-H4 mAb and anti-Ly6C or anti-NK1.1, respectively. H4^int thymic T cells express high levels of CD44, whereas H4^high thymic T cells express an intermediate amount of CD44 (Fig. 4A). Moreover, H4^high thymic T cells do not coexpress Ly6C or NK1.1 molecules at all (Fig. 4, B and C). Ly6C^high and NK1.1⁺ thymocytes correspond to H4^int thymic T cells (Fig. 4, B and C). Thus, H4^int thymic T cells are ordinary NKT cells, whereas H4^high thymic T cells constitute a specialized NKT-like T cell subpopulation that is not defined by the NKT phenotype of CD44^high, Ly6C^high, and NK1.1⁺.

V repertoire and sequences of TCR in Vß7⁺ H4^high and H4^low thymic T cells

Pairing with polyclonal Vß2⁺, -7⁺, and -8⁺ ß-chains, an invariant -chain composed of V14 and J281 is expressed in TCR of murine NKT cells (16, 17, 27). We examined whether the invariant -chain is expressed in NKT-like H4^high thymic T cells. In addition, it might be possible that H4^int and H4^high thymic T cells that escape negative selection in DBA/2 mice are oligoclones. Therefore, the repertoire of TCR- and ß-chains in Vß7⁺ H4^high thymic T cells in DBA/2 mice was analyzed and compared with that in Vß7⁺ H4^low thymic T cells.

T cell blasts selectively expressing Vß7 were induced from DBA/2 Vß7⁺ H4^high thymic T cells (Fig. 5A) and BALB/c Vß7⁺ H4^low thymic T cells (Fig. 5B) by in vitro stimulation with YPM. PCR of cDNA obtained from these T cell blasts using V and C primers indicated that V14 transcripts are predominantly expressed over the other V examined in H4^high thymic T cells (Fig. 5Ca), whereas the amount of V14 transcripts is decreased in H4^low thymic T cells (Fig. 5Cb). Furthermore, only one nucleotide insertion (data not shown) and J281 usage in all junctional regions resulted in identical amino acid sequences derived from V14⁺ -chains in Vß7⁺ H4^high thymic T cells (Fig. 5D). Both H4^low and H4^high thymic T cells revealed heterogeneous junctional sequences in the Vß7⁺ ß-chain, and the length of CDR3 region (the distance from the J region-encoded GXG triplet to the nearest preceding V region-encoded cysteine according to Rock et al. (28)) was diverse (Fig. 5, E and F). Thus, it is clear that invariant V14-J281⁺ thymic T cells (hereafter designated as V14⁺ thymic T cells) are enriched in H4^high thymic T cells, and those in DBA/2 mice do not consist of oligoclones. These results together with those in Fig. 4 indicate that V14⁺ thymic T cells consist of ordinary NKT cells and a new type of V14⁺ T cell.

View larger version (47K): [in this window] [in a new window]   FIGURE 5. Invariant V14-J281+ T cells are enriched in H4high thymic T cells. DBA/2 CD4 SP+DN thymocytes and BALB/c CD4 SP+DN thymocytes were obtained and sorted into Vß7+ H4high cells (A) and Vß7+ H4low cells (B), respectively. The sorted cells were stimulated with YPM in the presence of APCs followed by the expansion of blast cells with rIL-2. C, First-strand cDNAs obtained from YPM-induced DBA/2 (a) and BALB/c (b) thymic T cell blasts were amplified by PCR using various Vs and C primers. D, First-strand cDNAs derived from YPM-induced Vß7+ H4high thymic T cell blasts were amplified by PCR using V14 and C primers. Junctional sequences of V14+ -chain in cDNA clones were obtained as described in Materials and Methods. E and F, First-strand cDNAs derived from YPM-induced Vß7+ H4high or H4low thymic T cell blasts were amplified by PCR using Vß7 and Cß primers. Junctional sequences of Vß7+ ß-chain in cDNA clones were obtained as described in Materials and Methods.

Responsiveness of Vß7⁺ V14⁺ thymic T cells

Because Vß7⁺ T cells are potentially self-reactive in DBA/2 mice, it is of interest whether DBA/2 Vß7⁺ V14⁺ thymic T cells are responsive or anergic. In the experiment shown in Fig. 5, a substantial number of Vß7⁺ V14⁺ thymic T cell blasts was generated upon in vitro stimulation of DBA/2 H4^high thymic T cells with YPM, indicating that Vß7⁺ V14⁺ thymic T cells in DBA/2 mice are in an immunologically responsive state. We have previously found that CD1a^- CD4⁺ human thymic and cord blood T cells generated massive Vß2⁺ T cell blasts upon primary stimulation with TSST-1, and that those T cell blasts are in the anergic state upon restimulation with TSST-1. By contrast, Vß2⁺ CD4⁺ T cell blasts generated by stimulating human peripheral blood CD4⁺ T cells with TSST-1 exhibited enhanced IL-2 production upon restimulation with TSST-1 (29, 30). Thus, we consider that the responsiveness in this culture system can reflect the functional maturity of T cells. To further analyze the function of DBA/2 Vß7⁺ V14⁺ thymocytes, the primary and secondary responses upon stimulation with YPM were examined.

Vß7⁺ V14⁺ thymic T-enriched cells were obtained from BALB/c and DBA/2 CD4 SP+DN thymocytes by sequential immunomagnetic cell depletion and positive selection of H4⁺ cells containing mainly H4^int and H4^high thymic T cells (Fig. 6, Aa and Ab), and stimulated with YPM in the presence of APCs. BALB/c Vß7⁺ H4^int and H4^high thymic T cells produced a substantial amount of IL-2 (Fig. 6Ac) and IL-4 (Fig. 6Ad) upon stimulation with YPM. DBA/2 H4^int and H4^high thymic T cells had a much lower response than BALB/c H4^int and H4^high thymic T cells (Fig. 6, Ac and Ad). The lower proportion of TCR-ß^high H4^high thymic T cells in DBA/2 H4⁺ thymic T cells than BALB/c H4⁺ thymic T cells (Fig. 6, Aa and Ab) may be the cause of lower responsiveness. Alternatively, cells not triggered by the stimulation with YPM may be present in DBA/2 mice.

View larger version (33K): [in this window] [in a new window]   FIGURE 6. Responsiveness of Vß7+ V14+ thymic T cells. A, Vß7+ V14+ thymic T-enriched population was obtained by serial treatments as described in Materials and Methods. The purity of cells from BALB/c (a) and DBA/2 (b) after preparation was analyzed by flow cytometry. Numbers indicate the percentage of H4+ cells calculated by subtracting background staining. A total of 5 x 104 cells were cultured with titrated amounts of YPM in the presence of 2 x 105 syngenic APCs. After 2.5 days of culture, IL-2 production (c) and IL-4 production (d) were evaluated. Results are expressed as mean ± SD of triplicate cultures. B, In parallel with the above culture, 1 x 105 cells from BALB/c mice and 5 x 105 cells from DBA/2 mice were cultured with 3 µg/ml YPM in the presence of 5 x 105 syngenic APCs. Blast cells were purified after 3 days of culture, expanded with rIL-2 for 4 days, and used as YPM-induced thymic T cell blasts. The purity of YPM-induced thymic T cell blasts from BALB/c (a) and DBA/2 (b) was analyzed by flow cytometry and PCR using Vs and C primers. A total of 1 x 105 YPM-induced thymic T cell blasts were restimulated with titrated amounts of YPM in the presence of 2 x 105 syngenic APCs. After 24 h of culture, IL-2 (c) and IL-4 (d) production were evaluated as in A. Data shown are from one of three experiments which gave similar results.

Biological difference between V14⁺ thymic T cells and CD4 SP thymocytes differentiated via mainstream pathway

Next, we assessed the biological difference between V14⁺ thymic T cells and mainstream thymic T cells. Thymic T cell subpopulations have quite different sensitivities to steroid hormones (26, 31); SP thymocytes are relatively resistant, whereas DP thymocytes are sensitive. We examined the effect of treatment of mice with dexamethasone on H4^int and H4^high thymic T cells. The number of BALB/c thymic T cell subpopulations was plotted against dexamethasone dose (Fig. 7). Dexamethasone induced the deletion of H4^int and H4^high thymic T cells that was around 3- and 4-fold larger than that of CD4 SP thymocytes at 300 µg and 900 µg of dexamethasone, respectively. Thus, V14⁺ thymic T cells are relatively sensitive to dexamethasone compared to mainstream CD4 SP thymocytes.

View larger version (23K): [in this window] [in a new window]   FIGURE 7. V14+ thymic T cells are relatively sensitive to dexamethasone in vivo. Various doses of dexamethasone in ethanol/saline were injected into BALB/c mice s.c. Two days later, thymocytes were stained with combinations of FITC-anti-CD8 and PE-anti-CD4, or anti-TCR-ß/FITC-anti-hamster IgG and biotin-anti-H4 mAb/avidin-PE. Relative cell number of thymic T cell subpopulations were calculated. Results are expressed as the mean and SD of 4 mice/group. (*, p < 0.001 when compared with the relative cell number of CD4 SP thymocytes at each dose.) Data shown are typical results from one of three experiments.

View larger version (27K): [in this window] [in a new window]   FIGURE 8. Comparison of responsiveness between Vß7+ mainstream thymic T cells and Vß7+ V14+ thymic T cells. A total of 5 x 105 BALB/c Vß7+ mainstream thymic T-enriched cells obtained as described in Materials and Methods were cultured with 3 µg/ml YPM in the presence of 5 x 105 syngenic APCs. After expansion of blast cells with rIL-2, YPM-induced Vß7+ mainstream thymic T cell blasts were obtained. The purity of these cells (A) was analyzed as in Fig. 6. In parallel, BALB/c-derived YPM-induced Vß7+ V14+ thymic T cell blasts were obtained as in Fig. 6. The proportion of TCR-ß+ T cells and Vß7+ T cells in the preparation of YPM-induced blast cells was 55.3% and 55.5%, respectively, and PCR showed the preferential usage of V14 in their TCRs (data not shown). IL-2 (B) and IL-4 (C) productions of YPM-induced Vß7+ mainstream and Vß7+ V14+ thymic T cell blasts upon restimulation with YPM were analyzed as in Fig. 6. Data shown are from one of three experiments which gave similar results.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

It was shown that the NKT phenotype of CD44^high, Ly6C^high, and NK1.1⁺ corresponds to that of H4^int thymic T cells, whereas H4^high thymic T cells do not express a high amount of CD44, Ly6C, or NK1.1. Because H4^high thymic T cells express a high amount of TCR, whereas H4^int thymic T cells express a low amount of TCR (Figs. 1C and 2), these results are consistent with the notion that thymic NKT cells express a lower amount of TCR than mainstream thymic T cells (16, 19). However, more importantly, data presented here indicate that there is a specialized population in the thymus expressing a high amount of an invariant V14⁺ TCR that is associated with H4, but not with CD44, Ly6C, and NK1.1. Thus, V14⁺ thymic T cells consist of ordinary NKT cells and a new type of V14⁺ T cells. The reason that this type of V14⁺ T cell population has not been demonstrated to date is probably because V14⁺ NKT cells have been characterized mainly by using NK1.1⁺ C57BL/6 mice, and H4^high thymic T cells are present at much lower levels in C57BL/6 mice than BALB/c mice (Fig. 4). This new type of V14⁺ T cell raises several questions. Do they originate from a common differentiation pathway as NKT cells, indicating the different maturation steps of V14⁺ T cells, or from separate pathways? Are their activation requirements different from those of ordinary NKT cells? In peripheral lymphoid organs, how much do they occupy and how do they regulate immune responses? In BALB/c splenic CD4⁺ T cells, some cells (8%) express a substantial amount of H4 irrespective of Vß in their TCRs, suggesting that H4 is the surface marker on activated/memory CD4⁺ T cells. This fact makes it difficult to demonstrate V14⁺ T cells in the periphery with relation to H4. However, we are currently examining how much a new type of V14⁺ T cell is present in the periphery. Obviously, further experiments will be required to resolve these questions.

V14⁺ thymic T cells were shown to be biologically different from mainstream thymic T cells in several ways. First, V14⁺ thymic T cells escape the negative selection in the thymus of Mtv-7⁺ DBA/2 mice, whereas mainstream thymic T cells are deleted. Second, the pattern of lymphokine production is distinct. YPM-induced V14⁺ thymic T cell blasts produce markedly higher levels of IL-4 and lower levels of IL-2 than YPM-induced mainstream thymic T cell blasts upon in vitro restimulation with YPM, consistent with the findings of primary stimulation of NKT cells with anti-TCR-ß Abs (32, 33). Third, V14⁺ thymic T cells are relatively sensitive to dexamethasone compared with CD4 SP mainstream thymocytes. Tamada et al. (34) have recently reported the resistance of NKT cells in both spleen and liver to dexamethasone-induced apoptosis. There are some possible explanations for the distinct reactivity of V14⁺ thymic T cells to dexamethasone. Although NKT cell precursors are suggested to reside in the thymus (35, 36, 37), the sites for the generation of V14⁺ thymic T cells and peripheral NKT cells might be separate as suggested by a couple of reports (38, 39), leading to the different susceptibility to dexamethasone-induced apoptosis. Because the activation of cells induces the translocation of glucocorticoid receptor to the nucleus (40, 41), it is likely that the status of activation of cells is a determinant of susceptibility to apoptosis. From this viewpoint, V14⁺ thymic T cells may be more activated than peripheral NKT cells.

There were some findings concerning the function of V14⁺ thymic T cells. BALB/c Vß7⁺ V14⁺ thymic T cells responded well to YPM in both primary and secondary culture, suggesting that they are capable of responding upon rechallenge with Ags. DBA/2 Vß7⁺ V14⁺ thymic T cells that escaped the negative selection were shown to be in an immunologically responsive state. The lower response of Vß7⁺ V14⁺ thymic T cells from DBA/2 mice upon primary stimulation with YPM than that from BALB/c mice may be due to the presence of some anergized T cells under the influence of Mtv-7-encoded superantigens. Further experiments are required to clarify this. However, the same degree of response of YPM-induced Vß7⁺ V14⁺ thymic T cell blasts from DBA/2 and BALB/c mice was observed upon restimulation with YPM. This finding clearly indicates that Vß7⁺ V14⁺ thymic T cells in DBA/2 mice contain T cells as functionally mature as those in BALB/c mice. Therefore, the mechanism of escape of V14⁺ thymic T cells from the negative selection is important. Some possible mechanisms can be proposed. V14⁺ thymic T cells that develop in contact with CD1⁺ cortical thymocytes (16, 42, 43) may not encounter their deleting ligands on stromal cells. However, previous reports have shown that DN NKT cells are selected in the thymus of anti-HY TCR/Rag2^-/- mice (44) or in the presence of specific class I MHC Ag (45). Thus, we infer that V14⁺ thymic T cells develop by interaction with Mtv-7⁺ stromal cells. Alternatively, it is possible that H4 molecules affect TCR-mediated signaling in thymic selection. The interaction of H4 with unknown ligands may reduce the overall avidity of TCRs on Vß7⁺ and Vß8.1⁺ V14⁺ thymic T cells for Mtv-7-encoded superantigens, rescuing them from negative selection. Finally, the TCR structure may be involved. Because hypervariable region 4 of the Vß domain is crucial for the recognition of superantigens (46, 47), sequences of hypervariable region 4 in Vß7⁺ and Vß8.1⁺ ß-chain of V14⁺ thymic T cells should be analyzed. In addition, it is known that non-Vß TCR elements such as V repertoire and the combination of V and J usage influence T cell recognition of Mls-1^a (12, 13, 14). Furthermore, Mtv-7⁺ mice show that most, but not all, thymocytes with the relevant TCR Vß are deleted in the thymus (9, 10, 11, 12), and that TCR V repertoire in T cells surviving the negative selection is skewed (10, 11, 12, 15). Thus, skewed V repertoire or invariant -chain may affect the affinity of TCR for Mtv-7-encoded superantigens, decreasing it to below the range for negative selection. In the present study, besides the predominance of V14, the V repertoire in Vß7⁺ H4^high thymic T cells is different from that in Vß7⁺ mainstream thymic T cells. The use of V8 in the former and that of V11 in the latter was higher than other Vs (Figs. 5, 6, and 8), thus supporting this hypothesis.

An important question arises as to whether these mature Vß7⁺ and Vß8.1⁺ V14⁺ T cells readily react to Mtv-7-encoded super-antigens in the periphery, leading to the harmful response to the host? Because the DBA/2 mouse is not an autoimmune model mouse, it is apparent that immunological self-tolerance is maintained in the mouse. Thus, the above possibility is less likely. Some mechanisms can be suggested to explain self-tolerance of Vß7⁺ and Vß8.1⁺ V14⁺ T cells in DBA/2 mice. DBA/2 Vß7⁺ and Vß8.1⁺ V14⁺ T cells may be induced into a state of anergy after interaction with Mtv-7⁺ cells in the periphery. However, the responsiveness of DBA/2 Vß7⁺ V14⁺ thymic T cells to YPM in vitro suggests that the induction of anergy in these cells is less likely. An alternative mechanism, which we favor, is based upon the above-mentioned hypothesis that a distinct TCR structure causes the decreased affinity for Mtv-7-encoded superantigens. Vß7⁺ and Vß8.1⁺ V14⁺ T cells interact with Mtv-7-encoded superantigens in the peripheral lymphoid organs, but their signaling will not result in full activation, but survival as in the thymus. Thus, Vß7⁺ and Vß8.1⁺ V14⁺ T cells in DBA/2 mice seem to be regulated to not exceed triggering thresholds at the interaction with Mtv-7-encoded superantigens, while maintaining the responsiveness to natural ligands. The data presented here thus provide the basis for future work exploring the novel mechanisms of self-tolerance.

	Acknowledgments

 
We thank Drs. K. Tomonari, G. Suzuki, H. Nariuchi, and T. Nakayama for provision of mAbs. We also thank Hisako Yagi for technical help.

	Footnotes

 
¹ This work was supported in part by grants from the Ministry of Education, Science, Sports, and Culture of Japan, the Ministry of Public Welfare of Japan, and the Associazione Italiana Ricerca sul Cancro (Milan).

² Address correspondence and reprint requests to Dr. Junji Yagi, Department of Microbiology and Immunology, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. E-mail address:

³ Abbreviations used in this paper: SP, single positive; DP, double positive; Mtv, mouse mammary tumor virus; TSST-1, toxic shock syndrome toxin-1; YPM, Yersinia pseudotuberculosis-derived mitogen; DN, double negative.

Received for publication April 7, 1999. Accepted for publication July 2, 1999.

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