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CD4⁺ CD25⁺ Regulatory T Cell Repertoire Formation in Response to Varying Expression of a neo-Self-Antigen ¹

Wistar Institute, Philadelphia, PA 19104

	Abstract

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We have examined the development of self-peptide-specific CD4⁺CD25⁺ regulatory T cells in lineages of transgenic mice that express the influenza virus PR8 hemagglutinin (HA) under the control of several different promoters (HA transgenic mice). By mating these lineages with TS1-transgenic mice expressing a TCR that recognizes the major I-E^d-restricted determinant from HA (site 1 (S1)), we show that S1-specific T cells undergo selection to become CD4⁺CD25⁺ regulatory T cells in each of the lineages, although in varying numbers. In some lineages, S1-specific CD4⁺CD25⁺ regulatory T cells are highly abundant; indeed, TS1xHA-transgenic mice can contain as many S1-specific CD4⁺ T cells as are present in TS1 mice, which do not express the neo-self HA. In another lineage, however, S1-specific thymocytes are subjected to more extensive deletion and far fewer S1-specific CD4⁺CD25⁺ regulatory T cells accumulate in the periphery. We show that radioresistant stromal cells can direct both deletion and CD4⁺CD25⁺ regulatory T cell selection of S1-specific thymocytes. Interestingly, even though their numbers can vary, the S1-specific CD4⁺CD25⁺ regulatory T cells in all cases coexist with clonally related CD4⁺CD25^– T cells that lack regulatory function. These findings show that the formation of the CD4⁺CD25⁺ regulatory T cell repertoire is sensitive to variations in the expression of self-peptides.

	Introduction

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Athough it is well established that autoreactive thymocytes can be subject to deletion during their development, it is also clear that not all autoreactive CD4⁺ T cells are eliminated from the peripheral T cell repertoire and that additional mechanisms exist to regulate these cells. The generation of T cells with immunoregulatory properties is one of the ways in which potentially autoaggressive CD4⁺ T cells are controlled. The best characterized of these are CD4⁺CD25⁺ T cells, which were first identified in mice that developed organ-specific autoimmunity following neonatal thymectomy (reviewed in Refs. 1, 2). CD4⁺CD25⁺ T cells constitute 5–10% of the CD4⁺ T cells in normal mice and are able to suppress the autoimmune disease that develops in neonatally thymectomized mice (3). In efforts to determine how these cells are generated during the processes of immune repertoire formation, it was shown that CD4 single-positive (SP; ⁴ CD4⁺CD8^–) CD25⁺ thymocytes could also exert regulatory functions, providing evidence that regulatory T cells can be generated intrathymically (4, 5, 6). In addition, elegant studies using mice in which target organs such as the thyroid or ovaries had been removed showed that interactions with tissue-specific peptides in the periphery can also play a vital role in CD4⁺CD25⁺ regulatory T cell development or maintenance (7, 8, 9). When these tissues were removed from mice, the tissue-specific CD4⁺CD25⁺ regulatory T cells that are present in normal mice were not detectable (8, 10). Together, these studies suggested that processes occurring in both the thymus and the periphery can contribute to CD4⁺CD25⁺ regulatory T cell development, although the fate of autoreactive CD4⁺ T cells that recognize self-peptides could not be examined directly.

Recently, transgenic (Tg) mouse models have been developed in which CD4⁺CD25⁺ regulatory T cell formation can be examined in detail (11, 12, 13, 14, 15). TS1xHA28 mice were derived by mating Tg mice that express influenza virus hemagglutinin (HA) under the control of a SV40 promoter (HA28 mice) with Tg mice expressing a HA-specific MHC class II-restricted TCR specific for the major I-E^d-restricted determinant site 1 (S1; TS1 mice) (11, 16, 17, 18). TS1xHA28 mice contain large numbers of S1-specific CD4⁺ T cells; indeed, they contain as many S1-specific CD4⁺ T cells as are present in TS1 mice (11). However, unlike TS1 mice in which S1-specific CD4⁺CD25⁺ regulatory T cells are rare, 50% of the S1-specific CD4⁺ T cells in TS1xHA28 mice are CD25⁺ regulatory T cells (11, 12). We previously showed that interactions with the S1 peptide expressed by radioresistant thymic stromal cells induce the selection of S1-specific CD4 SP CD25⁺ thymocytes in TS1xHA28 mice (12). In addition, S1-specific CD4⁺ T cells were shown to expand in the periphery of HA28 mice in response to S1 peptide, but not in mice lacking the S1 peptide even under conditions of lymphopenia (19). In this report, we have generated additional lineages of Tg mice expressing the HA under the control of different promoters. One of these lineages contained large numbers of S1-specific CD4⁺CD25⁺ regulatory T cells, similar to our findings in TS1xHA28 mice. Using another lineage, we show that CD4⁺CD25⁺ regulatory T cell development can also occur when S1-specific thymocytes are subjected to more overt deletion than occurs in TS1xHA28 mice and can be accompanied by the accumulation of far fewer S1-specific CD4⁺CD25⁺ regulatory T cells in the periphery. Notably, even when the total numbers of S1-specific CD4⁺ T cells vary in the different lineages, the S1-specific CD4⁺CD25⁺ regulatory T cells in all cases coexist with similar numbers of S1-specific CD4⁺CD25^– T cells that lack regulatory function. Together, these studies provide insights into the impact that variations in the expression of self-Ags can exert on CD4⁺CD25⁺ regulatory T cell development.

	Materials and Methods

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Mice

PevHA mice were generated by linking DNA encoding the influenza virus PR8 HA to the human -globin locus-control region (generously provided by M. Antoniou, National Institute for Medical Research, London, U.K.) (20, 21). -myoHA mice contain PR8 HA linked to the -myosin H chain promoter (generously provided by J. Robbins, University of Cincinnati, Cincinnati, OH) (22). Following injection into BALB/c x C57BL/6 zygotes, PevHA- and -myoHA-Tg lines were established by successive backcrossing for at least 10 generations to BALB/c mice (Harlan Breeders, Indianapolis, IN) before use in this study. Transgene-encoded HA mRNA was detected by RT-PCR analysis in a variety of tissues as previously described (18). HA28 mice contain DNA encoding the NH₂-terminal 237 aa of PR8 HA linked to the SV40 early region promoter/enhancer elements and have been described elsewhere (17, 18). TS1 mice express a transgene-encoded TCR specific for S1, the major I-E^d-restricted determinant of PR8 HA, and have also been described previously (16). Mice were genotyped by PCR of excised tail DNA using HA- or TS1- specific primers as described elsewhere (16, 17). TS1^+/+ mice were mated with RAG2^–/– mice to generate TS1^+/–RAG^–/– mice (TS1.RAG). Mice were maintained in sterile microisolators at the Wistar Institute Animal Facility (Philadelphia, PA).

Flow cytometry

Flow cytometric analysis was performed on single-cell suspensions of 10⁶ cells prepared from pooled lymph nodes (LNs: inguinal, brachial, and axillary), from spleen, or from thymus. Three- and four-color analyses were performed on FACScan or FACSCalibur flow cytometers, respectively (BD Biosciences, San Hose, CA). Eighty thousand to 300,000 live events were collected according to forward/side scatter characteristics, and CellQuest software (BD Biosciences) was used to collect and analyze data. Purified populations were sorted at the Wistar Institute’s sorting facility on a Cytomation MoFlo using Summit Software (DakoCytomation, Fort Collins, CO). The following Abs were used for sorting and cell analysis and were purchased from BD PharMingen (San Diego, CA): allophycocyanin-anti-CD4, FITC-anti-CD4, FITC-anti-CD8, FITC-anti-CD25 (PC61 and 7D4), PE-anti-CD25 (PC61), and PE-anti-CD45RB. Biotin-6.5 was also used (16). Streptavidin-Red670 (Invitrogen Life Technologies, Rockville, MD) was used to detect biotinylated reagents.

CFSE labeling and adoptive transfer

LN cells purified by FACS from TS1xPevHA mice were labeled with CFSE (Molecular Probes, Eugene, OR) as previously described (23). LN cells were prepared as single-cell suspensions in serum-free supplemented IMDM (SF medium) medium at 37°C, incubated with 5 mM CFSE at 1 x 10⁷ cells/ml for 4–5 min, and then incubated with an equal volume of serum and washed with SF medium. Briefly, 0.5–1.5 x 10⁷ CFSE-labeled cells were resuspended in SF medium and adoptively transferred into the tail veins of PevHA or BALB/c recipients. Five days later, recipient LNs were harvested, stained with PE-anti-CD25, 6.5-biotin, and allophycocyanin-anti-CD4, and analyzed by flow cytometric analysis.

In vitro proliferation/suppression analysis

For proliferation assays, FACS-purified LN cells (2.5–5 x 10⁴ cells/well) were cultured with 5 x 10⁵ irradiated BALB/c splenocytes (APCs) and incubated with 0.3 or 1 µM S1 peptide (SFERFEIFPK; synthesized and HPLC purified by the Wistar Institute peptide synthesis) facility in 200 µl of supplemented IMDM containing 10% FBS in flat-bottom 96-well plates in the presence or absence of exogenous IL-2 (10 ng/ml) from the X-2 cell line (24). For suppression assays, FACS-purified cells from TS1xHA Tg mice were mixed with S1 peptide (0.3 or 1 µM), APCs, and unfractionated LN cells from TS1 mice (3.5–5 x 10⁴ cells/well) at varying suppressor:responder ratios. In each case, cells were pulsed with 0.5 µCi/well ³H-tritiated thymidine after 72 h and the amount of incorporated ³H-tritiated thymidine was determined 16 h later.

In vitro cytokine secretion analysis

LN cells from TS1 mice were coincubated with 1 µM S1 peptide, irradiated APCs, and FACS-purified CD25⁺CD4⁺ or CD25^–CD4⁺ LN cells as described above. After 48 h, one-half of the culture supernatant was harvested and the concentration of the indicated cytokines was measured by ELISA. mAb used for ELISA capture were: anti-IL-2 (JES5-1A12; BD PharMingen); anti-IFN- (RA-6A2; American Type Culture Collection), and anti-IL-3 (MP2-8F8; BD PharMingen); biotinylated mAb used for ELISA detection were: anti-IL2, (JES6-5H4; BD PharMingen), anti-IFN- (XMG1.2; American Type Culture Collection), and anti-IL-3 (MP2-43D11; BD PharMingen); cytokine standards used were: IL-2 (X-2; American Type Culture Collection, IFN- (19301T; BD PharMingen), and IL-3 (19221T; BD PharMingen).

Bone marrow (BM) cell preparation

Donor BM was harvested from TS1, TS1.RAG, TS1xPevHA, TS1xHA28, or TS1x-myoHA mice and depleted of T lymphocytes using either MACS columns or Dynal magnetic beads. For MACS columns, BM was stained with biotinylated Abs to CD4 and CD8 followed by incubation with streptavidin-conjugated magnetic beads and loaded onto MACS columns, and depletion was performed according to the manufacturer’s protocol (Miltenyi Biotec, Auburn, CA). For Dynal beads, BM was incubated with anti-CD4- and anti-CD8-conjugated Dynabeads and negative selection was performed using a Dynal MPC-L magnet according to the manufacturer’s protocol (Dynal, Lake Success, NY).

BM chimeras

BALB/c, PevHA, HA28, or -myoHA mice, 4–12 wk of age, were exposed to 900 rad of gamma irradiation, and reconstituted with 4–5 x 10⁶ T cell-depleted BM cells via tail vein injection 24 h later. Mice received neomycin sulfate (0.2 mg/ml) on the day of irradiation and every 2 days for 2–3 wk after irradiation and were analyzed 2–5 mo following reconstitution.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Different numbers of 6.5^highCD4⁺CD25⁺ regulatory T cells develop in TS1xPevHA and TS1x-myoHA mice

We have previously described selection events leading to the development of large numbers of self-peptide-specific CD4⁺CD25⁺ regulatory T cells in TS1xHA28 mice (11, 12, 19). TS1 mice express a Tg TCR that is specific for the major I-E^d-restricted determinant from HA (S1) and can be detected by the anti-clonotypic mAb 6.5 (16). HA28 mice express the PR8 HA as a transgene driven by the SV40 early region promoter/enhancer. We wanted to determine whether expression of HA under the control of different promoters might alter the selection of 6.5^highCD4⁺CD25⁺ regulatory T cells. This might indicate whether the abundant development of 6.5^highCD4⁺CD25⁺ regulatory T cells that occurs in TS1xHA28 mice is in some way unique or idiosyncratic to the HA28 lineage. It might also reveal how CD4⁺CD25⁺ regulatory T cell development can be affected by the diverse amounts and cell types in which self-peptides are expressed. Accordingly, two additional lineages of HA-Tg mice were generated. First, PevHA mice were generated by linking DNA encoding the PR8 HA to the human -globin locus-control region (20). Previous studies have shown that these sequences can direct expression of transgenes to erythroid lineage cells (21) and also to thymic epithelium, muscle, pancreatic islet cells and dendritic cell and macrophage precursors (25, 26, 27). Consistent with these previous studies, transgene-encoded HA mRNA was detected by RT-PCR in a variety of tissues from young adult PevHA mice, including the BM, spleen, and thymus (data not shown). Second, -myoHA mice were generated by linking DNA encoding the PR8 HA to the -myosin H chain promoter, which has previously been shown to direct expression of transgenes to the heart and smooth muscle fibers (22). RT-PCR analysis of RNA obtained from tissues of young adult -myoHA mice revealed HA mRNA in the heart and skeletal muscle, but it was also detectable in other tissues including the thymus, LNs, and spleen (data not shown). Overall then, PevHA and -myoHA mice exhibited somewhat diffuse HA mRNA expression patterns despite the putative tissue specificity of their promoters, and they were mated with TS1 mice to examine how 6.5^highCD4⁺ T cells developed relative to TS1 and TS1xHA28 mice.

TS1xPevHA mice contained similar numbers of 6.5^high CD4 SP thymocytes as TS1xHA28 mice, although in both cases the numbers of 6.5^high CD4 SP thymocytes were 25% lower than in TS1 mice (Fig. 1, A and C). The levels of 6.5, CD4, and CD3 were 40% lower on 6.5^high CD4 SP thymocytes from TS1xPevHA mice than from TS1 mice, as was previously described for 6.5^high CD4 SP thymocytes from TS1xHA28 mice (Fig. 1A and data not shown) (12). Furthermore, in both TS1xPevHA and TS1xHA28 mice, 25–30% of the 6.5^high CD4 SP thymocytes were CD25⁺, whereas <1% of the 6.5^high CD4 SP thymocytes were CD25⁺ in TS1 mice (Fig. 1A). By contrast, 6.5^high CD4 SP thymocytes were substantially less abundant in TS1x-myoHA mice than in TS1 mice; on average, TS1x-myoHA mice contained 10% as many 6.5^high CD4 SP thymocytes as TS1 mice. Despite their lower frequency, the percentage of 6.5^high CD4 SP thymocytes that was CD25⁺ in TS1x-myoHA mice was similar to TS1xPevHA and TS1xHA28 mice (38 vs 25–30%; Fig. 1, A and C). The 6.5^high CD4 SP thymocytes that were present in TS1x-myoHA mice also contained lower levels of 6.5 and CD4 than TS1 mice (Fig. 1A and data not shown), resembling the phenotype of 6.5^high CD4 SP CD25⁺ thymocytes that develop in TS1xHA28 and TS1xPevHA mice. When the LNs of the different lineages were examined, TS1xPevHA and TS1xHA28 mice were again found to contain strikingly similar frequencies of 6.5^highCD4⁺ T cells and in each case approximately one-half were CD25⁺CD45RB^int (Fig. 1, B and C). A much smaller percentage of 6.5^highCD4⁺ T cells in TS1 mice were CD25⁺CD45RB^int. Moreover, the development of these CD25⁺CD4⁺ LN cells in TS1 mice requires coexpression of endogenous TCR chains, because no 6.5^highCD4⁺CD25⁺ T cells develop in TS1.RAG mice (Ref. 12 and see Fig. 4).

View larger version (48K): [in this window] [in a new window]   FIGURE 1. 6.5highCD4+CD25+ regulatory T cell development in TS1xHA Tg mice. A, Staining of CD4 SP thymocytes with 6.5 was compared between TS1xPevHA, TS1xHA28, and TS1x-myoHA mice (thick lines) vs TS1 mice (thin lines). 6.5high cells were then analyzed for CD25 expression. The percentages of 6.5high or CD25+ cells (designated by brackets) are shown as average values ±SD (n 5). B, Histograms represent staining of 6.5 on CD4+ LN cells. Dot plots show CD25 vs CD45RB staining on 6.5highCD4+ LN cells. Overlays and averages as in A. The percentages of CD25+CD45RBint cells (designated by box) are shown as average values ± SD. C, Bar graph displays the mean numbers of CD4 SP 6.5high thymocytes and CD4+6.5high LN cells (pooled inguinal, brachial, and axillary) in each of the mice. Circles indicate values obtained from individual mice. D, CD4+CD25+ or CD4+CD25– T cells were purified from pooled LN cells of TS1xPevHA mice by FACS, labeled with CFSE, and 0.5–1.5 x 106 cells were injected i.v. into nonirradiated PevHA or BALB/c mice. Five days later, LN from recipient mice were harvested, pooled, and stained. Dot plots compare CFSE vs CD25 staining of CD4+6.5high LN cells and are representative of three experiments.

View larger version (43K): [in this window] [in a new window]   FIGURE 4. Radioresistant elements induce both deletion and CD25+ regulatory T cell selection of 6.5high CD4 SP thymocytes in TS1x-myoHA mice. TCR and CD25 levels are compared on TS1x-myoHA-BM chimeras (TS1-myoHA or TS1x-myoHABALB/c, thick line) vs TS1BALB/c chimeras (thin line) or on TS1.RAG-myoHA chimeras (thick line) vs TS1.RAGBALB/c chimeras (thin line). A and C, Histograms show expression of 6.5 on CD4 SP thymocytes and CD25 expression on 6.5high CD4 SP thymocytes. The percentages of 6.5high or CD25+ cells (designated by brackets) are shown as average values ± SD. B and D, Histograms show expression of 6.5 on CD4+ LN cells, and dot plots demonstrate the expression of CD25 vs CD45RB on 6.5highCD4+ LN cells. The percentages of 6.5highCD25+CD45RBint cells (designated by brackets or boxes) are shown as average values ± SD. FACS plots are representative of at least seven mice.

Unlike TS1xHA28 and TS1xPevHA mice, there appeared to be little peripheral expansion of 6.5^high CD4⁺ T cells in TS1x-myoHA mice. Indeed, whereas TS1xPevHA and TS1xHA28 mice contained 400% more 6.5^highCD4⁺ T cells than 6.5^highCD4⁺ SP thymocytes, there was no significant difference in the numbers of 6.5^high CD4 SP thymocytes and 6.5^highCD4⁺ T cells in TS1x-myoHA mice (as was also the case in TS1 mice; Fig. 1, B and C). Thus, in addition to containing fewer 6.5^high CD4 SP thymocytes, TS1x-myoHA mice also contained substantially fewer 6.5^highCD4⁺ LN cells than were present in TS1, TS1xPevHA, and TS1xHA28 mice. Interestingly, however, even though TS1x-myoHA mice contained many fewer 6.5^highCD4⁺ T cells than TS1xHA28 and TS1xPevHA mice, in this case also approximately one-half were CD25⁺CD45RB^int.

6.5^highCD4⁺CD25⁺, but not 6.5^highCD4⁺CD25^– T cells exhibit regulatory activity

We have previously shown that the CD4⁺CD25⁺ T cells from TS1xHA28 mice can suppress the in vitro proliferative responses of the CD4⁺CD25^– T cells with which they coexist (11). However, it was recently reported that both 6.5^highCD4⁺CD25⁺ and 6.5^highCD4⁺CD25^– T cells can exhibit regulatory activity when isolated from mice in which HA expression is driven by an Ig promoter (13). To more closely examine whether 6.5^highCD4⁺CD25^– T cells from TS1xHA28 mice exhibit regulatory activity, 6.5^highCD4⁺CD25⁺ and 6.5^highCD4⁺CD25^– T cells were purified from TS1xHA28 mice by cell sorting. The purified 6.5^highCD4⁺CD25^– T cells from TS1xHA28 mice exhibited comparable proliferation and secreted similar levels of IL-2, IL-3, and IFN- in response to S1 peptide to LN cells from TS1 mice (Fig. 2A). By contrast, 6.5^highCD4⁺CD25⁺ T cells exhibited much lower proliferative responses and reduced cytokine production. When mixed in equal numbers with LN cells from TS1 mice, the 6.5^highCD4⁺CD25⁺ cells also potently inhibited both proliferation and cytokine production. By contrast, addition of 6.5^highCD4⁺CD25^– T cells had no effect, indicating that these cells do not exhibit the ability to suppress CD4⁺ T cell responses in vitro.

View larger version (28K): [in this window] [in a new window]   FIGURE 2. 6.5highCD4+CD25+ T cells are hypoproliferative and inhibit CD4+CD25– effector T cell functions in vitro. A, Whole TS1 LN cells from TS1 mice were coincubated with S1 peptide and irradiated APCs along with either sorted CD4+CD25+ or CD4+CD25– LN cells from TS1xHA28 mice. Forty-eight hours later, one-half of the culture supernatant was harvested and the concentrations of the indicated cytokines were measured by ELISA. Cultures were replenished with medium and after another 24 h proliferation was determined. Data are representative of two independent experiments. B, Left panel, purified CD4+CD25+ or CD4+ CD25– LN cells from TS1xHA28 mice were stimulated with S1 peptide in the presence or absence of IL-2 and proliferation was determined based on [3H]thymidine uptake. Right panel, Whole LN cells from TS1 mice were incubated with purified CD4+CD25+ () or CD4+CD25– () cells from TS1xHA28 mice added at indicated suppressor cell:TS1 LN cell ratios, and proliferation was determined based on [3H]thymidine uptake. Values are the average ± SD and are representative of three experiments. C and D, Similar experiments as in B performed with 6.5+CD4+CD25+CD45RBint and 6.5+CD4+CD25–CD45RBhigh LN cells purified from TS1xPevHA and TS1xMyoHA mice, respectively.

Radioresistant elements dominate 6.5^highCD4⁺CD25⁺ regulatory T cell development in TS1xPevHA mice

To evaluate the potential involvement of different tissue elements in directing CD4⁺CD25⁺ regulatory T cell development, radiation BM chimeras were generated. The development of 6.5^highCD4⁺CD25⁺ T cells in PevHA mice reconstituted with TS1 BM (TS1PevHA mice) paralleled that in both TS1HA28 chimeras and intact TS1xPevHA mice (Figs. 3A and 1A). The 6.5^high CD4 SP thymocytes in both TS1PevHA and TS1HA28 mice expressed lower levels of 6.5 and CD4 than their TS1BALB/c counterparts (Fig. 3A and data not shown), and large fractions expressed CD25. The percentages of 6.5^highCD4⁺ LN cells that were CD25⁺CD45RB^int in TS1PevHA and TS1HA28 mice also closely resembled those of intact TS1xPevHA and TS1xHA28 mice (Figs. 3B and 1B). By contrast, 6.5^high CD4 SP CD25⁺ thymocytes were undetectable in TS1BALB/c mice or in TS1xHA28BALB/c or TS1xPevHABALB/c mice (Fig. 3A). The LN cells of TS1xPevHABALB/c mice contained a higher percentage of 6.5^highCD4⁺CD25⁺CD45RB^int T cells (18 ± 3%) than were present in TS1xHA28BALB/c or TS1BALB/c mice (8 ± 5% and 4 ± 2%, respectively), although this percentage was much lower than was present in TS1xPevHA or TS1PevHA mice (57 ± 10% and 65 ± 4%, respectively). The 6.5^highCD4⁺CD25⁺CD45RB^int T cells in TS1xPevHABALB/c may be regulatory T cells developing in response to BM-derived elements (as has been described previously (13)), although they may also be effector cells. Thus, although BM-derived elements may make a minor contribution, radioresistant elements in TS1xPevHA mice play a dominant role in inducing 6.5^high CD4 SP thymocytes to undergo selection to become CD25⁺ regulatory T cells, as previously described in TS1xHA28 mice.

View larger version (43K): [in this window] [in a new window]   FIGURE 3. Radioresistant elements dominate 6.5highCD4+CD25+ regulatory T cell development in TS1xPevHA mice. BALB/c, PevHA, or HA28 hosts were irradiated and reconstituted with T cell-depleted BM from TS1, TS1xPevHA, or TS1xHA28 mice, and mice were harvested 2–4 mo later. Cell surface markers are compared in BM chimeras that expressed HA in radioresistant cell types (TS1PevHA or TS1HA28) or BM-derived cells (TS1xPevHABALB/c and TS1xHA28BALB/c; thick lines) vs TS1BALB/c chimeras (thin line). A, Histograms show expression of 6.5 on CD4 SP thymocytes. 6.5high CD4 SP cells were then analyzed for CD25 expression. The percentages of 6.5high or CD25+ cells (designated by brackets) are shown as average values ±SD and are representative of seven mice in PevHA chimeras and of at least two mice in HA28 chimeras. B, Histograms represent expression of 6.5 on CD4+ LN cells. Dot plots compare CD25 vs CD45RB staining on 6.5highCD4+ LN cells. The percentages of 6.5high or CD25+CD45RBint cells (designated by brackets or boxes) are shown as average values ± SD and are representative of at least seven mice.

Radioresistant elements direct enhanced deletion and CD4⁺CD25⁺ T cell selection of the 6.5 TCR in TS1x-myoHA mice

We also generated BM chimeras to examine the contribution of different cell types to 6.5^highCD4⁺CD25⁺ T cell development in TS1x-myoHA mice. The frequency of 6.5^high CD4 SP thymocytes and of 6.5^highCD4⁺ LN cells in TS1-myoHA mice was substantially lower than in TS1BALB/c mice, and resembled intact TS1x-myoHA mice (Fig. 4, A and B). Despite the reduced frequency of 6.5^high cells in TS1-myoHA mice, large percentages of 6.5^high CD4 SP thymocytes and 6.5^highCD4⁺ LN cells were CD25⁺ and CD25⁺CD45RB^int, respectively, again closely paralleling intact TS1xMyoHA mice. By contrast, the development of 6.5^high CD4 SP thymocytes and 6.5^highCD4⁺ LN cells in TS1x-myoHABALB/c mice was very similar to TS1BALB/c mice (Fig. 4, A and B). Thus, despite their lower numbers relative to TS1xPevHA and TS1xHA28 mice, both deletion and 6.5^highCD4⁺CD25⁺ regulatory T cell selection are mediated by radioresistant elements in TS1x-myoHA mice, and BM-derived elements make little or no contribution to this process.

Because of their low frequency, we wanted to confirm that the 6.5^highCD25⁺ regulatory T cells that were detected in TS1x-myoHA mice had been generated through interactions between the 6.5 TCR and the S1 peptide. In particular, we wanted to exclude the possibility that enhanced deletion of thymocytes expressing high levels of the 6.5 TCR caused the residual 6.5^high CD4 SP thymocytes in TS1x-myoHA mice to contain elevated numbers of CD25⁺ thymocytes that coexpressed endogenous TCR -chains (4, 16, 28). Accordingly, additional chimeras were generated using BM obtained from TS1.RAG mice and irradiated -myoHA and BALB/c recipients. The percentage of 6.5^high CD4 SP thymocytes and CD4⁺ LN cells was reduced in TS1.RAG-myoHA mice compared with TS1.RAGBALB/c mice (Fig. 4, C and D). In these mice, 6.5^– CD4 SP thymocytes and 6.5^– CD4⁺ T cells must have derived from autoreconstitution by radioresistant CD4⁺ T cells from the recipient mice, and deletion of the 6.5 TCR allowed for more expansion of these cells in TS1.RAG-myoHA mice than in TS1.RAGBALB/c mice (Fig. 4C). Less than 1% of the 6.5^high CD4 SP thymocytes and 6.5^highCD4⁺ T cells in TS1.RAGBALB/c mice were CD25⁺ (Fig. 4C), consistent with previous studies showing that thymocytes that can only express the 6.5 TCR do not undergo CD25⁺ T cell selection in mice that lack the S1 peptide (12, 13). Significantly, however, the percentages of 6.5⁺ CD4 SP thymocytes that were CD25⁺ in TS1.RAG-myoHA mice were similar to the percentages in TS1-myoHA mice (Fig. 4, A and C). Likewise, the majority of the 6.5^highCD4⁺ LN cells in TS1.RAG-myoHA mice were CD25⁺CD45RB^int (Fig. 4D). Thus, thymocytes that only express the 6.5 TCR are subject to CD25⁺ T cell selection by the S1 peptide in -myoHA mice, even in the context of enhanced deletion mediated by radioresistant thymic stromal cells.

	Discussion

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The studies here examined the formation of CD4⁺CD25⁺ regulatory T cells expressing the 6.5 TCR in Tg mice expressing the HA under the control of three different promoters. TS1xPevHA and TS1xHA28 mice each contained high frequencies of 6.5^highCD4⁺CD25⁺ regulatory T cells despite using unrelated promoters to drive HA transgene expression, but TS1x-myoHA mice contained one-tenth as many 6.5^highCD4⁺CD25⁺ regulatory T cells as were present in the other lineages. Radioresistant thymic epithelial elements were responsible for both thymocyte deletion and regulatory T cell selection, since HA expression restricted to these elements recapitulated the 6.5^highCD4⁺CD25⁺ regulatory T cell development that occurred in intact TS1xHA Tg mice. Despite their varying numbers, the 6.5^highCD4⁺CD25⁺ regulatory T cells in each case coexisted with similar numbers of 6.5^highCD4⁺CD25^– T cells that failed to suppress the in vitro proliferation of S1-specific CD4⁺ T cells from TS1 mice. The studies provide evidence that the numbers of CD4⁺CD25⁺ regulatory T cells that are produced can be shaped by selection processes that occur in the thymus and by expansion in the periphery, and that these processes are sensitive to variations in self-peptide expression. They further suggest that CD4⁺CD25⁺ regulatory T cell selection can often be accompanied by the generation of clonally related CD4⁺CD25^– T cells that lack regulatory function.

A prominent feature of our previous studies in TS1xHA28 mice was the abundance of 6.5^highCD4⁺CD25⁺ regulatory T cells. Indeed, TS1xHA28 mice were found to contain as many 6.5^highCD4⁺ T cells as were found in TS1 mice (that do not express the S1 peptide), but in TS1xHA28 mice approximately one-half of these cells were CD25⁺ (11). In other reports using Tg mice to analyze CD25⁺ regulatory T cell development in response to self-peptides, CD25⁺ T cells expressing the clonotypic TCR under study were substantially less abundant than in TS1xHA28 mice (13). This raised the possibility that something idiosyncratic or peculiar about the expression of the S1 peptide in TS1xHA28 mice allowed for the formation of large numbers of 6.5^highCD4⁺CD25⁺ regulatory T cells. However, we have shown here that the total numbers of 6.5^highCD4⁺ SP thymocytes and 6.5^highCD4⁺ T cells, and the percentages that are CD25⁺, are equivalent in TS1xPevHA and TS1xHA28 mice. This striking similarity in 6.5^highCD4⁺CD25⁺ T cell development occurs even though different and unrelated promoters (the -globin locus control region vs the SV40 early region promoter/enhancer) direct HA expression in PevHA and HA28 mice. Moreover, the abundant development of 6.5^highCD4⁺CD25⁺ regulatory T cells in TS1xHA28 and TS1xPevHA mice is not determined by some unknown property of the 6.5 transgene. Far fewer 6.5^highCD4⁺CD25⁺ regulatory T cells develop in TS1x-myoHA mice, and this must be due to differences in the presentation of the S1 peptide between the mice. In the different TS1xHA Tg lineages analyzed here, the expression of S1 peptide under the control of different promoter/enhancer sequences could be permissive for the abundant formation of 6.5^highCD4⁺CD25⁺ regulatory T cells (in TS1xHA28 and TS1xPevHA mice), but could also lead to their development in much smaller numbers (in TS1x-myoHA mice). Based on these findings, we would propose that the repertoire of CD4⁺CD25⁺ T cells directed to self-peptides in non-Tg systems may similarly contain some abundant specificities and some that are more rare, depending at least partly on differences in how individual self-peptides are expressed.

Previous studies have shown that the thymus plays an important role in CD25⁺ regulatory T cell selection (12, 13, 29, 30, 31). However, thymocytes can also undergo deletion in response to self-peptides (28, 32, 33, 34, 35, 36) and the relationship between CD25⁺ T cell selection and deletion of autoreactive thymocytes remains poorly understood. In the studies here, TS1x-myoHA mice were found to contain significantly fewer 6.5^high CD4 SP thymocytes than TS1xPevHA and TS1xHA28 mice, although in all cases similar percentages (30%) were CD25⁺. Moreover, in all cases, the development of 6.5^highCD25⁺ thymocytes in BM chimeras in which HA expression was restricted to radioresistant thymic epithelial cells recapitulated their development in intact TS1xHA Tg mice. Self-peptides expressed by thymic epithelial cells have been shown to induce thymocytes to undergo selection to become CD25⁺ regulatory T cells (12, 13). The studies here extend these previous findings by showing that such selection can occur concurrently with deletion of autoreactive thymocytes. 6.5^high CD4 SP thymocytes might be subjected to enhanced deletion in TS1x-myoHA mice because the S1 peptide is expressed in higher amounts or by different subsets of thymic stromal cells than in TS1xPevHA or TS1xHA28 mice (37, 38). Alternately, the balance between CD25⁺ regulatory T cell selection and T cell deletion may be determined by expression of S1 by different subsets of thymic stromal cells (e.g., cortical vs medullary epithelium) (39, 40, 41, 42). It is clear, however, that the enhanced deletion of 6.5^high thymocytes in TS1x-myoHA mice was not mediated by BM-derived elements (31), because 6.5^highCD4⁺ T cell development was unaffected in TS1x-myoHABALB/c compared with TS1BALB/c chimeras. Instead, radioresistant thymic epithelial cells alone are able to mediate different degrees of deletion of autoreactive thymocytes in a process that can be accompanied by the generation of CD4⁺CD25⁺ regulatory T cells.

Events occurring in the periphery also appear to make a sizable contribution to the large numbers of 6.5^highCD4⁺ T cells that accumulate in TS1xPevHA and TS1xHA28 mice. Although TS1xPevHA and TS1xHA28 mice contained similar numbers of 6.5^highCD4⁺ T cells as TS1 mice, they contained only 25% as many 6.5^high CD4 SP thymocytes. For similar numbers of 6.5^highCD4⁺ T cells to accumulate from significantly fewer 6.5^high CD4 SP thymocyte precursors implies that 6.5^high cells underwent more expansion in the periphery of TS1xPevHA and TS1xHA28 mice than in TS1 mice. Indeed, we recently showed that 6.5^highCD4⁺CD25⁺ T cells proliferate upon transfer into nonirradiated HA28 mice, but not in irradiated BALB/c recipients (19). Similarly, we showed here that both 6.5^highCD4⁺CD25⁺ and 6.5^highCD4⁺CD25^– T cells from TS1xPevHA mice undergo proliferation when reintroduced into the periphery of nonlymphopenic PevHA mice. In addition, the percentages of cells that were CD25⁺ increased from 30% of 6.5^high CD4 SP thymocytes to 50% of 6.5^highCD4⁺ T cells in the periphery of TS1xPevHA and TS1xHA28 mice. By contrast, there was a much smaller increase in the numbers of 6.5^highCD4⁺ cells and in the percentages that were CD25⁺ between CD4 SP thymocytes and CD4⁺ T cells in TS1x-myoHA mice. Together, these observations suggest that differences in the expression of the S1 peptide affect the numbers of 6.5^highCD4⁺CD25⁺ regulatory T cells that accumulate in the periphery of the different lineages. Future experiments will determine how these differences in S1 expression affect the proliferation of CD25⁺ T cells, their sensitivity to apoptosis, or conversions that may occur between CD25⁺ and CD25^– subsets (30, 43, 44).

It was also noteworthy that a minor population of 6.5^highCD4⁺CD25⁺ T cells could be detected in the periphery of TS1xPevHABALB/c mice, even though 6.5^high CD4 SP CD25⁺ thymocytes were undetectable in these chimeras. It is possible that these cells are regulatory T cells that are generated in the periphery in response to BM-derived cells (13). However, we found little evidence that 6.5^highCD4⁺CD25^– T cells undergo conversion into 6.5^highCD4⁺CD25⁺ T cells following adoptive transfer into nonlymphopenic PevHA hosts, even though they went through multiple rounds of division. This argues against the de novo formation of 6.5^highCD4⁺CD25⁺ T cells in the periphery of TS1xPevHA mice, at least under the conditions studied here. It is also possible that these cells represent effector cells; indeed, such effector cells might develop because 6.5^highCD4⁺CD25⁺ regulatory T cell development is driven by expression of self-peptides on thymic epithelial cells, and does not occur in these chimeras. Ongoing studies are examining whether the 6.5^highCD4⁺CD25⁺ T cells that develop in TS1xPevHABALB/c mice exhibit comparable regulatory activity in vitro and/or express similar levels of Foxp3 mRNA (45, 46, 47) on a per cell basis to those that develop in TS1PevHA mice. Although these studies may reveal a role for BM cells in 6.5^highCD4⁺CD25⁺ T cell development, it is also clear that radioresistant elements play a dominant role in the development of 6.5^highCD4⁺CD25⁺ T cells in all of the lineages described here. In each case, BM chimeras expressing HA only on radioresistant cells closely resembled intact mice.

Finally, it is significant that 6.5^highCD4⁺ T cells exist as roughly equal mixtures of CD25⁺ and CD25^– T cells in all of the lineages of TS1xHA Tg mice described here. Moreover, whereas the 6.5^highCD4⁺CD25⁺ cells suppressed the in vitro proliferation and cytokine secretion by CD4⁺ T cells from TS1 mice in response to the S1 peptide, the 6.5^highCD4⁺CD25^– T cells exhibited no suppressive activity. We also showed that purified 6.5^highCD4⁺CD25^– T cells from all three lineages proliferate in response to the S1 peptide. This contrasts with findings when HA expression is driven by the Ig promoter (13), in which both 6.5^highCD4⁺CD25⁺ and 6.5^highCD4⁺CD25^– T cells were found to be anergic and regulatory. However, the findings here are consistent with many studies conducted in non-Tg systems showing that the immune repertoire contains pathologic autoreactive CD25^– T cells whose activity is prevented by regulatory CD4⁺CD25⁺ T cells, and that these populations can include cells that share the same tissue specificity (7, 8, 9, 29). By using several different promoters to drive HA expression in HA Tg mice, we provide evidence that the generation of CD4⁺CD25⁺ regulatory T cells may typically be accompanied by the development of clonally related CD25^– T cells bearing an identical TCR specificity, even when they are produced in different total numbers. These CD25^– T cells appear to be both more proliferative than their CD25⁺ counterparts and unable to suppress T cell proliferation in vitro, and it will be important in future studies to more fully define the properties of and relationships between these subsets.

	Acknowledgments

 
We thank A. E. Hollenbeck for excellent experimental assistance, H. M. Guay and A. Rankin for thoughtful discussion, and J. S. Faust and A. Acosta at the Wistar Institute Flow Cytometry Facility for cell sorting. PevHA and -myoHA mice were generated under the supervision of Dr. Jean Richa at the Transgenic Mouse Facility, University of Pennsylvania School of Medicine.

	Footnotes

 
¹ This study was supported by grants from the National Institutes of Health, from the Lupus Foundation of Southeastern Pennsylvania, and by the Commonwealth Universal Research Enhancement Program, Pennsylvania Department of Health.

² Current address: Signal Transduction Laboratory, Abrahamson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA 19104.

³ Address correspondence and reprint requests to Dr. Andrew J. Caton, Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104. E-mail address: caton{at}wistar.upenn.edu

⁴ Abbreviations used in this paper: SP, single positive; HA, hemagglutinin; BM, bone marrow; LN, lymph node; S1, site 1; Tg, transgenic; SF, serum free; int, intermediate.

Received for publication June 18, 2003. Accepted for publication April 22, 2004.

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