Development of CD25+ T cells secreting transforming growth factor-ß1 by altered peptide ligands expressed as self-antigens
Hiromichi Yamashiro1,
Nobumichi Hozumi1 and
Naoko Nakano1
1 Research Institute for Biological Sciences, Science University of Tokyo, 2669 Yamazaki, Noda City, Chiba 278-0022, Japan
Correspondence to: N. Nakano; E-mail: naoko{at}rs.noda.sut.ac.jp
Transmitting editor: K. Inaba
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Abstract
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This study demonstrates that CD4+ T cells specific for an altered self-antigen differentiate to T cells secreting transforming growth factor (TGF)-ß1. In this study, we utilized mice expressing an altered peptide ligand containing a single amino acid substitution of moth cytochrome c 88103 peptide. In these mice, antigen-specific T cells escaping thymic negative selection differentiated into T cells with an effector/memory phenotype, CD44high, CD45RBlow, CD62L and CD25intermediate. The expression of CD25 and high levels of CD44 was initiated in the thymus during the development from CD4+CD8+ to CD4+; a large proportion of maturing CD4+ thymocytes expressed both CD25 and high levels of CD44. Upon antigen stimulation, CD4+ T cells derived from these mice did not proliferate or secrete IL-2, but secreted TGF-ß1. Neutralizing antibodies to TGF-ß1 reversed the impaired proliferative responses to the antigen, suggesting that TGF-ß1 secreted from these T cells negatively regulates T cell responses.
Keywords: altered peptide ligands, self-antigen, transforming growth factor-ß1
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Introduction
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The immune system is required to have a broad repertoire to cope with various kinds of antigens, while immune responses against self-antigens have to be avoided. T cells with all different specificities are placed in an environment where the interactions of a TCR with self-peptideMHC can control homeostatic balance. A pivotal mechanism inducing tolerance is the deletion of autoreactive T cells in the thymus (negative selection) (13). However, as thymic tolerance is never complete, autoreactive T cells are either anergized or suppressed in the periphery (46). Furthermore, these autoreactive T cells can actively suppress autoimmune responses (7).
Engagement of a TCR with an antigen peptideMHC complex leads to rapid phosphorylation of signaling molecules and increase of intracellular Ca2+ levels, resulting in cellular proliferation and the expression of cytokine genes. Altered peptide ligands (APL), which have amino acid substitutions, can either inhibit agonist responses (antagonists) or have only some functions (partial agonists) (810). Recognition of APL by the TCR induces downstream signaling events with qualitative and/or quantitative differences from signaling induced by the parent peptide (1012). Differing kinetics of binding, determined by the off-rate of the interaction between the TCR and the peptideMHC complex, may generate these distinct signals (1316). Some APL induce distinct patterns of TCR
chain phosphorylation (17,18), correlating with the ability to induce distinct signals. The C-terminal portion of cytochrome c is a well-characterized antigenic peptide binding to MHC class II I-E molecules. In normal mice, T cells specific for the C-terminal portion of cytochrome c use Vß3 and V
11 almost exclusively (19). Dissection of TCR binding to the moth cytochrome c 88103 (MCC) peptide demonstrated that positions 99 and 102 of the MCC peptide interact with the CDR3 regions of TCR
and ß respectively (20).
Transforming growth factor (TGF)-ß exerts a variety of activities in different tissues (21). The regulatory mechanisms of TGF-ß in the immune system are important to maintain tolerance (22). TGF-ß1 knockout mice develop severe inflammatory diseases (23), suggesting that TGF-ß1 expression suppresses autoimmune responses. TGF-ß1 is involved in oral tolerance (24); following administration with oral antigen, isolated T cells secrete TGF-ß1 that regulates autoimmunity (25). T cell clones secreting TGF-ß1 in addition to IL-5 and IL-10 inhibit antigen-specific T cell responses (26,27). Studies of mice expressing a dominant negative form of the TGF-ß type II receptor within T cells demonstrated that TGF-ß1 plays an important role in the homeostatic balance of the immune system (28,29).
In this study, we examined tolerized T cells being interacted with an APL expressed as a neo-self-antigen. We generated transgenic mice expressing an APL containing a single amino acid substitution at either position 99 or 102 (99E or 102E) of MCC. In mice expressing 99E or 102E, MCC-specific T cells differentiated into T cells secreting TGF-ß1. Development of these T cells took place in the thymus, with the induction of CD25 and CD44 expression during the differentiation from CD4+CD8+ to CD4+ cells. Our results demonstrate that relatively low affinity interactions between TCR and self-peptides induce the development of T cells secreting TGF-ß1.
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Methods
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Transgenic mice
Expression vectors for fusion proteins of MCC88103 or its APL, 99E and 102E, with invariant chain (Ii) p31 have been reported previously (30). The EcoRIBglII fragment encoding Ii-peptide was introduced into the ß-globin expression cassette of pDOI-5 (31). A BglI fragment, including the MHC class II E
promoter and the ß-globin expression cassette, was purified and injected into (C57BL/SJL)F2 fertilized eggs. Founder mice, identified by Southern blotting, were backcrossed onto the B10.BR background for >6 generations. Transgene expression was measured by S1 mapping; 1020 µg RNA was hybridized to a probe containing part of the vector and Ii sequence. The level of transgene expression was then compared to the endogenous Ii expression. AND mice transgenic for the TCR specific for pigeon cytochrome c peptide (32) were purchased from the Jackson Laboratories (Bar Harbor, ME). B10.BR mice were obtained from Sankyo (Tokyo, Japan). All the mice were bred on the B10.BR background under pathogen-free conditions.
Immunization of mice and T cell proliferation assays
All peptides (>90% purity) (MCC: ANERADLIAYLKQATK; 99E: ANERADLIAYLEQATK; 102E: ANERADLIAYLKQAEK) were purchased from Sawady Technology (Tokyo, Japan). Mice (710 weeks old) mice were injected in the footpads with either 100 µg peptide or PBS (as a control) in complete Freunds adjuvant (Difco, Detroit, MI). Draining lymph nodes were obtained 9 days after immunization; isolated cells (3 x 105/well) were stimulated with varying concentrations of peptide in 200 µl complete medium. TGF-ß1 was neutralized using anti-TGF-ß1 antibodies (R & D Systems, Minneapolis, MN; AB-101-NA). At 72 h after stimulation, cells were pulsed with 1 µCi [3H]thymidine for 18 h and the incorporation of [3H]thymidine was measured.
Antigen presentation assay
To analyze the expression of Ii-peptide, we measured the proliferation of MCC-, 99E- or 102E-reactive CD4+ T cells isolated from immunized B10.BR mice by a MACS microbeads column (Miltenyi Biotech, Sunnyvale, CA). Varying quantities of irradiated (30 Gy) splenocytes from Ii-peptide transgenic mice or those from B10.BR mice with (1 and 0.1 µM) or without peptide were cultured with antigen reactive CD4+ T cells (1 x 105/well). After 72 h, cells were pulsed for 18 h with 1 µCi [3H]thymidine to measure the incorporation of [3H]thymidine.
Semiquantitative RT-PCR
Total RNA was isolated from cells with Isogen-TRIzol (Wako, Osaka, Japan). cDNAs were then synthesized from 5 µg total RNA using a RT-PCR kit (Stratagene, Cedar Creek, TX). Then 10% of the cDNA was amplified with various sets of primers obtained from Amersham Pharmacia Biotech (Arlington Heights, IL). The gene encoding the housekeeping enzyme, HPRT, was used as a control. The sequences of 5' and 3' primers were IL-2: 5'-TGATGGACCTACAGGAGCTCCTGAG-3', 5'-GTTCTAGAACAGTTACTCTGATATTGC-3', TGF-ß1: 5'- AGATCTCCCTCGGACCTGCTGGCAGT-3', 5'-CACGGCACT TCGGAGAGCGGGAAC-3', HPRT: 5'-GTAATGATCAGTCAA CGGGGGAC-3', 5'-CCAGCAAGCTTGCAACCTTAACCA-3'. Thirty cycles of amplification were performed at 94°C for 45 s for denaturing, 55°C for 45 s for annealing and then 72°C for 1 min to extend. Conditions were optimized to generate PCR products in a linear range.
Purification of naive and effector/memory T cells
Naive CD4+ T cells from AND mice were isolated by serial purification with MACS beads. Following staining of total spleen cells with FITCanti-CD4 (PharMingen, San Diego, CA), cells were positively selected on an anti-FITC-conjugated MACS beads column. Bound beads to the cells were then cleaved according to the manufactures protocol. Isolated cells were stained with FITCanti-CD62 ligand (CD62L) (PharMingen) and re-applied to a fresh anti-FITCconjugated MACS beads column. The cells that did not bind to the second column were designated as effector/memory (CD4+CD62L) cells; those bound were eluted as the naive (CD4+CD62L+) T cell population. The purity of each subset of CD4+ T cells was
9095%.
ELISA
Cytokine levels were determined by ELISA. Antibodies used in the capture and detection (biotin-labeled) were purchased from PharMingen (18161D and 18172D for IL-2; 23201D and 23212D for TGF-ß1). Recombinant IL-2 was purchased from Peprotech (Rocky Hill, NJ), TGF-ß1 was obtained from Genzyme/Techne (Minneapolis, MN). Alkaline phosphatase-conjugated streptavidin (Vector, Burlingame, CA) was used in the second step of detection. The total amount of TGF-ß1 (active + latent) was measured by heating at 80°C for 10 min to convert into active form before the assay. The pre-existing active form was determined without the heat treatment.
Flow cytometry
Cells derived from the lymph node or the thymus were stained with various combinations of PEanti-CD4 (L3T4; Becton Dickinson, Mountain View, CA), FITCanti-CD4 (L3T4; PharMingen), FITCanti-CD8 (Ly-2; Becton Dickinson), CyChromeanti-CD8 (Ly-2; PharMingen), CyChromeanti-CD44 (IM-7; PharMingen), FITCanti-CD62L (MEL-14; PharMingen), biotinanti-CD45RB (16A; PharMingen) and biotinanti-CD25 (7D4; PharMingen). PEstreptavidin (Gibco/BRL, Gaithersburg, MD) was used to detect biotinylated antibodies. Staining of Vß3 and V
11 was performed using culture supernatants of specific hybridoma (KJ25 and RR8.1 respectively), followed by FITCgoat anti-hamster IgG (Caltag, Burlingame, CA) and biotingoat anti-rat IgG (Southern Biotechnology Associates, Birmingham, AL) respectively. Red670streptavidin (Gibco/BRL) was used for the last step. PEanti-V
11 (RR-8.1; PharMingen) was also used when specified. Following three washes, cells were analyzed on a FACSort (Becton Dickinson) using CellQuest software. Data are presented on log scale as either dot-plots or histograms.
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Results
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Generation of transgenic mice
To generate mice expressing MCC and its APL as a neo-self-antigen, sequences coding for the 88103 peptide of MCC, or its 99E and 102E analogs, were introduced into the C-terminal portion of Ii (30) (Fig. 1A). These fusion proteins were expressed under the control of the MHC class II E
promoter (31). We chose the substitution of K at position 99 with E (99E) or of T at position 102 with E (102E), which introduces a dramatic change in the interaction of the peptideMHC with the TCR
and ß respectively (20). Neither 99E nor 102E have agonistic effects on the bulk of MCC-reactive T cells induced in vivo or have any antagonistic activity on the MCC-specific hybridomas tested (data not shown). These constructs were used to generate transgenic mice, from which we selected the Ii-MCC-37G, Ii-99E-10G and Ii-102E-29 lines (called Ii-MCC, Ii-99E and Ii-102E hereafter), expressing the transgenes at similar levels. The expression levels were tested by S1 mapping to distinguish the endogenous Ii transcript from the transgenic Ii-peptide mRNA. The ratios of Ii-peptide transgene expression to the endogenous Ii expression in the thymus were 0.19 for Ii-MCC, 0.17 for Ii-99E and 0.25 for Ii-102E (data not shown). Expression in the spleen was similar to that in the thymus. To detect the expression of these transgenes as a peptideMHC complex, we measured the ability of transgenic splenocytes to stimulate T cells reactive against the corresponding peptides. Responses were compared to those elicited by normal splenic APC pulsed with graded doses of peptides. In all three cases, stimulation was roughly equivalent to that induced by APC pulsed with 0.1 µM peptide (Fig. 1B), with slightly higher stimulation by the Ii-102E splenocytes.

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Fig. 1. Expression of Ii-peptide molecules. (A) Structures of Ii-peptide molecules expressed by transgenes. Sequences of MCC, 99E and 102E peptides with a cathepsin D cleavage site (cath. site) were added to the C-terminal portion of Ii p31. (B) Increasing numbers of splenocytes (irradiated) either from Ii-MCC, Ii-99E or Ii-102E transgenic mice (solid lines with closed circles) were cultured with antigen-reactive CD4+T cells (1 x 105/well) and proliferation was measured. Spleen cells from B10.BR mice pulsed with either 1 µM peptide (dotted lines with open triangles), 0.1 µM peptide (dotted lines with open squares) or no peptide (dotted lines with open circles) were used as controls.
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TGF-ß1 expression was induced in MCC-specific T cells in transgenic mice expressing Ii-99E and Ii-102E
We analyzed the responses of T cells against MCC in transgenic mice expressing either Ii-MCC, Ii-99E or Ii-102E. Ii-peptide mice in a B10.BR background and non-transgenic B10.BR mice were immunized with MCC and antigen-specific proliferative responses of draining lymph node cells were measured in vitro. As shown in Fig. 2(A), the responses to MCC were completely blocked in the mice expressing Ii-MCC. Expression of either 99E or 102E also strongly suppressed responses to MCC. The impaired responses to MCC in Ii-99E and Ii-102E mice may result from either the deletion of MCC-reactive T cells in the thymus or the unresponsiveness of surviving cells exported in the periphery. To examine the reaction of peripheral T cells to MCC in mice expressing 99E and 102E, we performed RT-PCR to detect cytokines expressed in cells stimulated by MCC. Following the re-stimulation of cells with MCC for 8 h in vitro, the expression of IL-2 was observed in cells from non-transgenic control animals, while no IL-2 expression was detected in cells from Ii-99E and Ii-102E mice (Fig. 2B). Cells from Ii-99E and Ii-102E mice, however, expressed elevated levels of TGF-ß1 even without in vitro re-stimulation. Enhanced induction of TGF-ß1 was also seen during in vivo priming in Ii-102E. Moreover, cells obtained from immunized Ii-102E mice expressed TGF-ß1 earlier than control animals, by 2 days after immunization, continuing until day 11 (Fig. 2C). These results suggest that MCC-reactive T cells in Ii-102E mice may have been committed to express TGF-ß1 prior to encountering the antigen.

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Fig. 2. TGF-ß1 is expressed in response to MCC in mice expressing 99E or 102E. (A) Ii-MCC, Ii-99E and Ii-102E mice were immunized with MCC. Draining lymph node cells (3 x 105/well) were cultured with varying concentrations of MCC and proliferation was measured. Closed circles with solid lines represent the responses in Ii-peptide transgenic mice; open circles with dotted lines designate the responses in wild-type (B10.BR) mice. (B) Total RNA was extracted from draining lymph node cells obtained from Ii-99E, Ii-102E and non-transgenic littermates as in (A) with or without in vitro MCC re-stimulation for 8 h. RT-PCR was performed with specific primers. HPRT was used as a control. (C) The expression of TGF-ß1 in Ii-102E and non-transgenic littermates was analyzed by RT-PCR at varying time points after immunization.
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AND T cells differentiated into T cells with effector/memory phenotype in Ii-99E and Ii-102E mice
To analyze precisely the development of MCC-reactive T cells expressing TGF-ß1 in Ii-99E or Ii-102E, we made use of TCR transgenic mice, AND, whose T cells are specific for the C-terminal fragment of pigeon cytochrome c (32). As expected, MCC peptide induced proliferation in AND T cells strongly. On the other hand, 102E did not stimulate T cells at all. Another APL, 99E, was able to induce proliferation at high concentrations (Fig. 3A). Then, AND mice were crossed with Ii-peptide mice to generate double-transgenic mice. Development of AND thymocytes in the mice expressing MCC, 99E or 102E were analyzed by FACS (Fig. 3B). AND thymocytes skewed toward CD4+ cells, as previously reported (32). In contrast, the thymi of AND/Ii-MCC and AND/Ii-99E mice contained both CD4 and CD8 single-positive cells with an increased percentage of double-negative thymocytes. The numbers of thymocytes in AND/Ii-MCC (4.6 ± 2.1 x 106, n = 3) and AND/Ii-99E (4.9 ± 1.4 x 106, n = 3) mice were reduced to one eighth that of Ii-peptide-negative AND littermates (38.7 ± 5.8 x 106, n = 4), indicating that most of AND T cells were negatively selected in the presence of Ii-MCC and Ii-99E. AND/Ii-102E mice, however, had
3 times more thymocytes (12.0 ± 2.9 x 106, n = 5) than AND/Ii-99E mice. A large proportion of the thymocytes in AND/Ii-102E mice differentiated into CD4+CD8+, but the differentiation to CD4+ was partially blocked. These results indicated that more stimulatory ligands deleted more AND thymocytes. The number of CD4+ T cells in the periphery reflected different efficiency of thymic selection. We could detect more CD4+ T cells in the spleens (data not shown) and the lymph nodes in AND/Ii-102E than in AND/Ii-99E mice, and the lowest in AND/Ii-MCC (Fig. 3C). The absolute numbers of peripheral CD4+ T cells in AND/Ii-99E mice were one-fifth that seen in AND mice. Although AND/Ii-MCC mice also had some CD4+ T cells in the periphery, the expression of transgenic TCR, V
11 and Vß3, was profoundly reduced. In comparison, although the levels of V
11 and Vß3 were down-regulated, they remained still higher in AND/Ii-99E and AND/Ii-102E mice (Fig. 4A). These results indicated the different affinity of peptide ligands primarily affected thymic selection of AND T cells and then controlled the expression levels of transgenic TCR. Interestingly, the majority of the peripheral CD4+ T cells in AND/Ii-MCC, AND/Ii-99E mice and a large portion of those in AND/Ii-102E mice did not display the phenotype of naive T cells any more. They had an effector/memory phenotype, having lost the expression of CD62L and expressing high levels of CD44 (Fig. 4B). These T cells also expressed low levels of CD45RB (Fig. 4C) and intermediate levels of CD25 (Fig. 4D).

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Fig. 3. The expression of MCC, 99E and 102E affects AND T cell development in vivo. (A) Proliferative responses of AND splenocytes (3 x 105 /wells) to MCC, 99E and 102E. (B) Thymocytes from 12-week-old AND, AND/Ii-MCC, AND/Ii-99E and AND/Ii-102E mice were stained with PEanti-CD4 and FITCanti-CD8, and analyzed by flow cytometry. (C) Lymph nodes cells from 12-week-old AND, AND/Ii-MCC, AND/Ii-99E and AND/Ii-102E mice were stained with FITCanti-CD4. The expression of CD4 is shown in a histogram.
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The expression of CD25 and high levels of CD44 was induced in CD4+CD8dull thymocytes by APL
Next, we examined where CD4+ T cells acquired an effector/memory phenotype. CD4+CD25+ regulatory T cells are reported to develop in the thymus (33) and are induced with self-antigens during the development in the thymus (34). Thymocytes from AND and AND/Ii-102E mice were analyzed for CD25 (Fig. 5B) and CD44 (Fig. 5C) expression in gated fractions, CD4+CD8+ (population I), CD4+CD8dull (population II) and CD4+ (population III) (Fig. 5A). The proportion of CD25+ cells significantly increased as thymocytes differentiated from population I to II in AND/Ii-102E mice; this increase was less in AND thymocytes. Although the proportion of CD25+ cells then decreased upon differentiation into CD4+ (population III), 23.8% of CD4+ thymocytes retained expression of CD25 in AND/Ii-102E mice, whereas only 0.71% expressed CD25 in AND mice. Similarly, the proportion of CD44high cells increased following differentiation to population II from I in AND and AND/Ii-102E mice. CD44 expression increased dramatically (41%) as the cells differentiate into population III in AND/Ii-102E mice, while decreasing to 1.1% in AND mice. AND/Ii-99E mice had few cells in population I and II; however, 3-day-old AND/Ii-99E mice had these populations, allowing us to determine that CD4+CD25+ thymocytes developed similarly to those in AND/Ii-102E (data not shown). As CD4+CD25+ thymocytes in TCR transgenic mice are reported to use endogenous
chains (33), the expression of V
11 in CD4+ thymocytes was analyzed after depletion of CD8-expressing thymocytes. V
11+CD4+ thymocytes were gated and analyzed for the expression of CD25 and CD44. V
11+CD4+ thymocytes in AND mice expressed minimal CD25 and low levels of CD44; however, a large proportion of those cells in AND/Ii-102E mice expressed CD25 and high levels of CD44 (Fig. 6). These results demonstrated that 99E and 102E, APL containing a single amino acid substitution of MCC, induced the development of CD4+CD25+CD44high AND T cells in the thymus.

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Fig. 5. CD4+ thymocytes in AND/Ii-102E mice express CD25 and high levels of CD44. (A) Thymocytes from both 12-week-old AND/Ii-102E and their littermates (AND/) were stained with either FITCanti-CD4, CyChromeanti-CD8 and biotinanti-CD25/Red670streptavidin or PEanti-CD4, FITCanti-CD8 and CyChromeanti-CD44. Expression of CD4 and CD8 (FITCanti-CD4 and CyChrome-anti-CD8) is shown and the gated populations (I, II and III) are indicated. (B and C) Expression of CD25 (B) or CD44 (C) was analyzed throughout the development from population I to II, then to III. Background staining is indicated by a gray line.
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T cells in AND/Ii-99E and AND/Ii-102E mice were anergic, secreting TGF-ß1 upon antigen stimulation
The phenotype of T cells developed in AND/Ii-99E and AND/Ii-102E mice was similar to that of activated T cells. To characterize these T cells, spleen cells from AND, AND/Ii-99E and AND/Ii-102E mice were stimulated with MCC for 24 h, and the production of IL-2 was measured. IL-2 was not detected at all in cultures of AND/Ii-99E and AND/Ii-102E splenocytes, whereas AND T cells produced a large amount of IL-2 (Fig. 7A). Proliferative responses to MCC were measured in the presence or absence of 50 U/ml murine recombinant IL-2. T cells in AND/Ii-99E and AND/Ii-102E splenocytes did not proliferate upon MCC stimulation; those impaired responses, however, were restored by the addition of exogenous IL-2, reaching response levels observed in AND T cells (Fig. 7B). To exclude the possibility that 99E and 102E expressed in APC inhibit responses, we isolated CD4+ T cells with an effector/memory phenotype (CD4+CD62L) from AND/Ii-99E mice. The proliferative responses of these cells to MCC presented by APC from B10.BR mice were compared to those of naive T cells (CD4+CD62L+) isolated from AND mice. CD4+CD62L T cells from AND/Ii-99E mice did not proliferate efficiently in response to antigen, while AND naive (CD4+CD62L+) T cells responded vigorously (Fig. 7C). Importantly, T cells of AND/Ii-99E and AND/Ii-102E, but not AND, mice secreted TGF-ß1 in response to even 0.1 µM MCC. As much as one-fourth to one-half of the total TGF-ß1 was active in culture (Fig. 8A). Neutralizing antibodies to TGF-ß1 partially rescued the suppressed responses to MCC from AND/Ii-99E and AND/Ii-102E splenocytes (Fig. 8B). Furthermore, the antigen-specific activation of AND CD4+ T cells analyzed by forward scatter (Fig. 8C) and IL-2 production (Fig. 8D) was suppressed by addition of AND/Ii-99E CD4+ T cells. Therefore, MCC-reactive T cells that developed in mice expressing either 99E or 102E become anergic T cells secreting TGF-ß1 upon antigen stimulation, which may lead to the suppression of antigen-specific T cell responses.

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Fig. 7. T cells in AND/Ii-99E and AND/Ii-102E mice are anergic. (A) Production of IL-2 was measured by ELISA in culture supernatants of splenocytes from AND, AND/Ii-99E and AND/Ii-102E mice, following stimulation with 1 µM MCC for 24 h. (B) Proliferation of splenocytes (3 x 105/well) from AND (circles), AND/Ii-99E (triangles) and AND/Ii-102E (squares) mice to varying concentrations of MCC was measured in the presence (open symbols and dotted lines) or absence (closed symbols with solid lines) of 50 U/ml IL-2. (C) Naive (CD62L+ CD4+) T cells from AND mice and effector (CD62L CD4+) T cells from AND/Ii-99E mice were sorted by MACS. The proliferation of CD4+ T cells (5 x 104/well) to MCC was measured in the presence of irradiated T-cell-depleted splenocytes (5 x 104/well) from B10.BR mice.
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Fig. 8. Antigen-specific induction of TGF-ß1 in T cells from AND/Ii-99E and AND/Ii-102E mice. (A) Active (solid bars) and latent (bars with stripes) forms of TGF-ß1 in culture supernatants of splenocytes from AND, AND/Ii-99E or AND/Ii-102E mice were measured by ELISA. Cells were cultured with (0.1, 0.3 or 1.0 µM) or without MCC for 24 h. (B) Proliferative responses of splenocytes from AND, AND/Ii-99E and AND/Ii-102E mice to MCC were measured in the presence (closed circles) or absence (open circles) of neutralizing antibodies against TGF-ß1 (1 ng/ml). Data are shown as the average ± SD. (C) Spleen cells from AND, AND/Ii-99E or AND + AND/Ii-99E (1:1 mixture as CD4+ cells) were cultured for 72 h with or without 1 µM MCC. Cells were stained with FITCanti-CD4, CD4+ cells are gated and the forward scatter height is shown. (D) The supernatant was obtained in 24 h from each culture shown in (C). Production of IL-2 was measured by ELISA. Data are shown as the average ± SD.
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Discussion
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In this study, we demonstrated that self-antigens with lower affinities to a TCR could induce the development of a distinct population of T cells regulating the immune response. In transgenic mice expressing the MCC peptide analogue, 99E or 102E, the proliferative responses of T cells were significantly suppressed and these T cells expressed TGF-ß1 abundantly in response to MCC. Development of such T cells was also clearly demonstrated in double-transgenic mice expressing AND TCR and 99E or 102E. Substantial numbers of CD4+ T cells were observed in the double-transgenic mice although AND T cells were largely deleted in the thymus. These T cells had an effector/memory phenotype, expressing high levels of CD44 and intermediate levels of CD25. They could neither express IL-2 nor proliferate upon antigen stimulation; however, antigen-induced proliferation could be restored by addition of exogenous IL-2, indicating that they were anergic and functionally different from normal effector T cells. Our observation with the mice expressing APL may be similar to previous reports (7,34), showing that the generation of anergic T cells could be induced by low-avidity interaction between a TCR and a self-antigen. The anergic CD4+T cells in AND/Ii-99E and AND/Ii-102E mice also secreted a large amount of TGF-ß1 to suppress additional T cell responses.
In the thymus of AND/Ii-102E mice, a large proportion of CD4+V
11+ thymocytes expressed CD25 and high levels of CD44 whereas those thymocytes in AND mice expressed almost no CD25 and low levels of CD44. CD4+CD8dull and CD4+ thymocytes already exhibited expression of CD25 and high levels of CD44. These observations indicated that MCC-specific thymocytes were selected to become CD25+CD44high T cells by the APL expressed in the thymus. The APL we used poorly induced proliferation in AND T cells. In fact, the substitution of a positively charged K at MCC position 99 to a negatively charged E or of T to E at position 102 has been reported to interfere the interaction of the peptideMHC complex with MCC-specific TCR (20). In AND/Ii-102E mice, a significant number of CD4+ T cells in the periphery expressed CD25 and high levels of CD44, although approximately half of the peripheral CD4+ T cells possessed a naive phenotype (CD62L+ CD25CD44low). In comparison, most of CD4+ T cells in AND/Ii-99E were CD62LCD25+CD44high, indicating that 99E was more efficient in inducing the development of CD25+CD44high cells than 102E. However, the 99E peptide, which is a more stimulatory ligand than 102E for the AND TCR, also deleted more AND T cells at the double-positive stage in the thymus. The most stimulatory ligand, the MCC peptide, deleted most of AND thymocytes; however, a very few AND T cells expressing CD25 and high levels of CD44 were still allowed to emigrate into the periphery. These T cells in AND/Ii-MCC also secreted TGF-ß1 upon antigen stimulation (data not shown). Thus, it appears that even the expression of MCC can induce the differentiation of AND T cells secreting TGF-ß1. Therefore, our double-transgenic mice expressing AND TCR and its ligand clearly demonstrated that the thymic negative selection and generation of CD25+CD44high thymocytes occur sequentially to establish self-tolerance. Jordan et al. demonstrated that the selection of CD4+CD25+ thymocytes can result from high affinity interaction with self-peptide (36). In their double-transgenic mice expressing a self-antigen and the antigen-specific-TCR, CD4+CD25+ thymocytes were selected without thymic deletion, while low-affinity interaction of a self-peptide with a TCR did not induce CD4+CD25+ thymocytes. Although we do not have a clear explanation for this discrepancy, in our transgenic mice expressing the peptide as a chimeric molecule with Ii chain, the self-peptide may be presented more efficiently by APC, which results in not only inducing CD4+CD25+ thymocytes but also deleting the T cells with the self-antigens expressed.
It has been reported that T cells with dual TCR can use one TCR for the positive selection and the other for the antigen recognition (37), raising the possibility that endogenous TCR were used for the selection of AND T cells expressing CD25 and high levels of CD44. CD25+CD44high thymocytes expressing both V
11 and CD4 developed in double-transgenic mice expressing AND TCR and the APL, but not in AND single-transgenic mice, indicating that CD25+CD44high thymocytes expressing transgenic TCR
chain are selected by the APL. Furthermore, the ability of the APL to stimulate AND T cells correlated well with their ability to induce CD25+CD44high CD4+ T cells. It thus appears that the interaction between AND TCR and the APL is responsible for these T cells. However, we cannot exclude the possibility that endogenous TCR collaborate with AND TCR to select CD25+CD44high CD4+ T cells. Therefore, to address this possibility, it will be required to analyze the development of CD25+CD44high thymocytes in a RAG-1/2-deficient background.
CD4+ T cells which have developed in AND/Ii-99E and AND/Ii-102E displayed several characteristics of regulatory T cells including expression of CD25, production of TGF-ß1 and being anergy. TGF-ß1, one of the major inhibitory cytokines, is known to be produced by some subsets of regulatory T cells (27,38). The suppressive mechanism of CD4+CD25+ regulatory T cells largely involves direct cellcell contact (33,39); however, there is also evidence that TGF-ß1 secreted by these T cells plays a role in suppression of autoreactive T cells. Administration of anti-TGF-ß1 antibodies can abrogate the prevention of Th1-cell-induced colitis by CD4+CD25+ regulatory T cells (40). Moreover, it has been shown that membrane-bound TGF-ß1 is expressed on CD4+CD25+ regulatory T cells (41), which can explain the requirement of cell contact and the suppressive role of TGF-ß1. It is noticeable that cytotoxic T lymphocyte-associated antigen (CTLA)-4 expressed by CD4+CD25+ regulatory T cells plays an essential role for the suppression (42). The signals induced by the ligation of CTLA-4 are also known to be required for induction of peripheral tolerance (43). Ligation of CTLA-4 has been shown to induce TGF-ß1 expression in CD4+ T cells (44). It is also noteworthy that the secretion of TGF-ß1 from CD4+ T cells in AND/Ii-99E and AND/Ii-102E mice was enhanced significantly by ligating CTLA-4 with anti-CTLA-4 antibodies in addition to anti-CD3 and anti-CD28 antibodies in our system (data not shown). The addition of neutralizing antibodies to TGF-ß1-producing cultures augmented the responses to MCC. However, TGF-ß1 may not be sufficient to mediate full suppression, as neutralizing antibodies to TGF-ß1 could only partially restore the AND T cell activation suppressed by CD4+ T cells from AND/Ii-99E mice (data not shown). Therefore, in addition to TGF-ß1, cellcell contact may also be required for suppression by CD4+ T cells in AND/Ii-99E and AND/Ii-102E mice. It will be of interest to further analyze whether these T cells suppress autoreactive responses in vivo.
In summary, this work demonstrated the development of antigen-specific T cells secreting TGF-ß1 in mice expressing an APL. The development of these T cells was induced by low-affinity interactions of TCR with self-antigens in the thymus. It could be possible that weak interactions of T cells with self-peptides generate T cells that secrete TGF-ß1 to keep a homeostatic balance in the immune system.
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Acknowledgements
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We are grateful to Ms M. Lemeur for the production of the transgenic lines, Drs D. Mathis and C. Benoist for critical reading of the manuscript, and Mr K. Hayashi for establishing the transgenic lines. This work was supported by the Ministry of Education, Science, Sport and Culture of Japan.
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Abbreviations
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APLaltered peptide ligand
CD62LCD62 ligand
CTLA-4cytotoxic T lymphocyte-associated antigen-4
Iiinvariant chain
MCCmoth cytochrome c 88103
TGFtransforming growth factor
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