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Cutting Edge: Size and Diversity of CD4⁺CD25^high Foxp3⁺ Regulatory T Cell Repertoire in Humans: Evidence for Similarities and Partial Overlapping with CD4⁺CD25^– T Cells¹

Nicolas Fazilleau^2,*, Hervé Bachelez, Marie-Lise Gougeon^* and Manuelle Viguier^2,*

^* Unité de Recherche et d’Expertise Immunité Anti-virale, Biothérapie et Vaccins, Institut National de la Santé et de la Recherche Médicale (INSERM) U668, Institut Pasteur; and INSERM U697, Université Paris 7, Hôpital Saint-Louis. Paris, France

	Abstract

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Both differentiation and function of CD4⁺CD25^high naturally arising regulatory T cells (Treg), which play a key role in the control of autoimmunity, are thought to depend on TCR specificity. In the present study, we comparatively measured the TCR repertoire sizes of human peripheral blood Treg and CD4⁺CD25^– T cells by using a methodology based on PCR amplification and sequencing analysis. We show that Treg use a large unrestricted TCR repertoire, the size and diversity of which are closely similar to those of CD4⁺CD25^– T cells, with a mean estimated size of 3.5 x 10⁶ distinct TCR vs 4.7 x 10⁶ distinct TCR for CD4⁺CD25^– T cells. In addition, a 24% overlap between the repertoires of these two CD4⁺ subsets in the periphery is found. These data emphasize the intersection between naturally occurring Treg and effector T cell peripheral repertoires and provide new insights into the ontogeny of Treg in humans.

	Introduction

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Thymus-derived CD4⁺CD25^high regulatory T cells (Treg)³ showing immunosuppressive functions have been recently described (1). These cells are characterized by the expression of Foxp3, a transcription factor, the expression of which is mandatory for their immunoregulatory function (2). Their critical role in immune tolerance has been demonstrated by studies showing that mutations of X-linked Foxp3 gene lead to a severe polyautoimmune syndrome (3). In mice, studies have shown that Treg develop mainly in the thymus and are positively selected after interactions between their TCR and self-peptides complexed with MHC molecules at the surface of stromal cells (4). The recent identification of few Ag recognized by Treg suggests that in vivo expansion of these cells could result from Ag-specific stimuli, although their immunosuppressive activity is exerted in a nonspecific manner (5). An important missing piece is the lack of large-scale in vivo studies analyzing the diversity of the TCR repertoire of Treg in humans. To date, preliminary studies suggest that Treg use TCR V segments at a similar frequency as CD4⁺CD25^– T cells and that the distribution of the various V CDR3 lengths is polyclonal, arguing for a wide repertoire and consequently for a large Ag specificity (6, 7). In the present study, we comparatively measured the TCR repertoire sizes of human peripheral Treg and CD4⁺CD25^– T cells by using PCR amplification and sequencing analysis. Using this method, we previously estimated the size of the repertoire of human T cells (8) and murine T splenocytes from wild-type mice and various deficient mice (9, 10). We demonstrate, herein, that Treg use a large unrestricted TCR repertoire, the size of which is closely similar to that of CD4⁺CD25^– T cells. We also show evidence for an overlap between the respective repertoires of CD4⁺ T cells.

	Materials and Methods

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Patients and samples

Blood samples (400 ml) were obtained from healthy donors after written informed consent was given. PBMC were isolated by Ficoll-gradient.

Cell purification

CD4⁺ PBMC were isolated using magnetic microbeads (Miltenyi Biotec) and labeled with FITC anti-CD4 (clone S3.5) or allophycocyanin anti-CD4 (clone SK3; BD Pharmingen) and PE anti-CD25 Ab (clone M-A251, BD Pharmingen). CD4⁺CD25^high and CD4⁺CD25^– cells were purified using a MoFlo cell sorter (DakoCytomation). Using FITC anti-V12.1 (clone 6D6.6; Pierce/Endogen), we isolated CD4⁺CD25^highV12.1⁺, CD4⁺CD25^highV12.1^–, and CD4⁺CD25^–V12.1⁺ T cells. The percentage of V12.1 chain usage was analyzed on a FACSCalibur flow cytometer using the CellQuest software (BD Biosciences).

FoxP3 expression

RNA from sorted cells was extracted using the RNeasy kit (Qiagen) and reverse transcribed into cDNA using oligo(dT) and SuperScript II (Invitrogen Life Technologies). Quantitative PCR using specific primers for FoxP3 and human hypoxanthine ribosyltransferase 1 was conducted as described previously (11).

Calculation of V usage and immunoscope analysis

Quantitative PCR amplifications were performed in a final volume of 25 µl on one twenty-fifth of the cDNA from sorted cells with TaqMan Universal PCR Master Mix (Applied Biosystems) with one of the 24 V gene segment-specific oligonucleotides, the C-specific antisense primer, and a fluorescent probe specific for the C gene segment as described elsewhere (11, 12). Amplified products were used as a template for an elongation reaction with a fluorescence-tagged oligonucleotide (12) and fluorescent products were loaded on a denaturating polyacrylamide gel and analyzed using immunoscope software (13).

Calculation of J gene segment usage

A single PCR amplification was conducted on cDNA using one given sense V-specific primer and the C-specific antisense primer. PCR were conducted with 5 U of Pfu polymerase (Stratagene) in the supplier’s buffer with the following cycling conditions: 10 min at 95°C, 30 cycles of 45 s at 95°C, 45 s at 60°C, and 1 min at 72°C. The PCR products were diluted (10^–6) and used as a template for quantitative PCR with one given V-specific primer, each of 13 TCRJ subfamily-specific primers, and a nested fluorochrome-labeled TaqMan probe for TCR V (5'-FAM-TTTGTCGGTACCAGTAAAG-3' for V6a and 5'-FAM-CATCTCCCTTATCAGTCACCTCAACATTCAT-TAMRA-3' for V14).

Cloning and sequencing of TCR V-J rearrangements

V-J PCR products were cloned in a pCR4Blunt-TOPO vector (Invitrogen Life Technologies). Sequencing reactions were performed using the ABI PRISM BigDye terminator reaction kit (Applied Biosystem). Reaction mixtures were analyzed on a 48-capillary 3730 DNA Analyzer (Applied Biosystems).

Calculation of the size of the TCR repertoire

The equation by Barth (14) and Behlke (15) was used to estimate the maximum number of distinct CDR3 sequences present in the cDNA of a given V-J rearrangement (maximum likelihood estimate (MLE)). The method used for repertoire size calculation has been reported (8, 9) as follows: size of the V repertoire = number of distinct sequences present in all CDR3 peaks/(frequency of V x frequency of J segments); size of the whole TCR repertoire = number of distinct sequences present in all CDR3 peaks from the V12.1⁺ T cells/(frequency of V x frequency of J segments x frequency of V12.1⁺ T cells in the studied population).

	Results and Discussion

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Suppressive activity and FoxP3 expression of isolated CD4⁺CD25^high T cells

The aim of this study was to accurately determine the size of the TCR repertoire from peripheral human Treg. One major caveat with Treg remains the lack of a specific cell surface marker, which hampers the purification of these cells. To date, the most specific marker of Treg is FoxP3 (2), which cannot be helpful for the isolation of these cells because of its exclusive intracellular expression. Cell surface markers for Treg have been described, such as the expression of CD25 (the IL-2 receptor -chain), the glucocorticoid-induced TNF receptor family-related protein (GITR), CTLA-4 molecules, or the down-regulation of IL-7 receptor (CD127) (16). However, high expression of CD25 is widely considered as a main marker of Treg, allowing the provision of a highly enriched population of Treg (1, 17). Therefore, we sorted CD4⁺CD25^high T cells from the PBMC of three healthy donors. Purity rates of CD4⁺CD25^high and CD4⁺CD25^– T cells were 96.9 ± 4.3% and 96.6 ± 3.3% (mean ± SD), respectively. We tested the activity of sorted CD4⁺CD25^high T cells in an in vitro allogeneic stimulation assay (11) and found that these cells were anergic and could inhibit the proliferation of autologous CD4⁺CD25^– T cells in a dose-dependent manner (data not shown). A high level of FoxP3 transcription was detected by quantitative RT-PCR in CD4⁺CD25^high T cells when compared with CD4⁺CD25^– T cells with a relative expression of 564 ± 86 and 2.9 ± 0.8, respectively (Student’s t test; p < 0.003). Altogether, we conclude that sorted CD4⁺CD25^high T cells display markers and regulatory properties characteristic of naturally occurring Treg.

The TCR V repertoire of human peripheral Treg is polyclonal

We first estimated the usage of each V gene segment in sorted T cells (Fig. 1A). We did not find statistically significant differences in V usage between Treg and CD4⁺CD25^– T cells (paired Student’s t test; p = 0.38). Analysis of CDR3 length distribution for all V-C rearrangements showed in both subsets nonbiased profiles, the hallmark of a polyclonal T cell repertoire (Fig. 1B). Altogether, these data confirm that both CD4⁺ populations express a highly diverse set of V subfamilies.

View larger version (30K): [in this window] [in a new window]   FIGURE 1. V and J gene segment usage in CD4+CD25high and CD4+CD25– T cells. A, Real-time PCR amplification analysis of the V usage by CD4+CD25high and CD4+CD25– T cells showing a similar V usage between the two populations. Mean percentages from three different donors are presented. B, CDR3 length distributions for CD4+CD25high and CD4+CD25– V6a-C and V14-C rearrangements from HD2 showing Gaussian distribution. The peak of the 10th codon is marked on the abscissa axis. C, Real-time PCR amplifications analysis of J usage by sorted CD4+CD25high and CD4+CD25– T cells. A representative experiment performed in triplicate is shown for V6a PCR products from HD2, showing a similar J usage between the two CD4+ populations. D, CDR3 length distributions for CD4+CD25high and CD4+CD25– V6a-J2.4 and V14-J2.7 rearrangements from HD2, showing Gaussian distribution. The peak of the 10th codon is marked on the abscissa axis.

TCR V repertoire size of human peripheral Treg

Various V-J combinations of sorted populations from two healthy donors (HD) were amplified, cloned, and sequenced. To limit bias, we chose V-J rearrangements with similar low usage frequencies in both populations (Table I) and used a statistical analysis (MLE) (8, 9) to estimate the number of distinct nucleotide sequences in the studied rearrangements. For example, 95 distinct sequences were found for the V6a-J2.6 combination in the Treg population from HD1 whereas a MLE value of 101 distinct sequences was calculated. Given that 2.34% of Treg bore a TCR using the V6a gene segment and that 0.52% of these V6a cells used the J2.6 gene segment, the TCR V repertoire size was estimated as follows: MLE x 1/percentage of V-J usage = 101 x 1/(2.34% x 0.52%) = 0.83 x 10⁶. Extending our study to another donor and to two other V-J rearrangements, we estimated that at least 0.24–1.16 x 10⁶ various V-chains were used by Treg at a given time (mean, 0.74 x 10⁶; median, 0.83 x 10⁶). Regarding CD4⁺CD25^– T cells, we estimated that 0.78 x 10⁶ various V-chains were used on average (median, 0.81 x 10⁶) (Table I). In conclusion, we show that the size of the TCR V repertoire is closely similar in Treg and CD4⁺CD25^– T cells.

View this table: [in this window] [in a new window]   Table I. Estimation of the TCRV repertoire size in human peripheral CD4+CD25high and CD4+CD25– T cells

The present data and those reported by Hsieh et al. (20) do not suggest that the pool of peripheral Treg is shaped by a restricted set of ligands but rather that the Treg repertoire is selected in the thymus by a large panel of self MHC class II-peptide complexes. However, these findings do not rule out the contribution of Ag-specific mechanisms in the peripheral expansion and maintenance of Treg clones following exposure to peptides derived from infectious agents or tumors as previously reported (21).

Size of the TCR repertoire of human peripheral Treg

A unique TCR V-chain associates on average with two to three distinct V-chains in mice (9) and 25–100 in humans (8). Even if TCR V-chain diversity is very similar in Treg and CD4⁺CD25^– T cells, one could hypothesize that pairing is different between the two subsets, leading to a different level of TCR repertoire diversity. To address this possibility, we sorted out CD4⁺CD25^highV12.1⁺, CD4⁺CD25^highV12.1^–, and CD4⁺CD25^–V12.1⁺ T cells from a third donor. We analyzed CDR3 length distribution and observed nonbiased curves for each V-C rearrangement. We next sequenced all CDR3 lengths of two different V-J combinations. Given the low number of CD4⁺CD25^highV12.1⁺ T cells recovered and the low frequency for each V-J combination, the number of distinct sequences was very low (Table II). Because the TCR repertoire of CD4⁺CD25^highV12.1⁺ T cells was polyclonal, the same estimation model was used. We estimated the TCR V repertoire size in this T cell subset to range from 0.056 to 0.113 x 10⁶ (Table II). Moreover, the minimal number of distinct TCR expressed by these cells, which corresponds to the TCR V diversity divided by the percentage of cells using V12.1 gene segments, was estimated to be on average 3.49 x 10⁶. We then evaluated the TCR diversity of CD4⁺CD25^– T cells to be on average 4.7 x 10⁶, indicating that the two CD4⁺ subsets share a similar level of TCR repertoire diversity (Table II). Comparative analysis with the estimated repertoire diversity of peripheral human CD4⁺ and CD8⁺ T cells indicates that TCR diversity in Treg is of same order of magnitude (8). When V12.1⁺ and V12.1^– CD4⁺CD25^high T cells were compiled, the estimated TCR V-chain diversity was 0.0115 to 0.067 x 10⁶ (Table III). The pairing, which corresponds to the TCR size divided by the compiled TCR V, was at least 72.8–182.6 for Treg (Table III), i.e., very close to estimations performed in total blood T cells (25–100) (8).

View this table: [in this window] [in a new window]   Table II. Estimation of the TCR repertoire size in human peripheral CD4+CD25high and CD4+CD25– T cells

View this table: [in this window] [in a new window]   Table III. Estimation of pairing in human peripheral CD4+CD25high T cells

These different observations argue for a smaller clone size in Treg than in CD4⁺CD25^– T cells. It remains to be determined whether this reflects lower avidity of Treg, TCR-ligand interactions, or TCR-independent mechanisms. Treg are able to suppress activated T cells in vitro at a ratio of one Treg to 20 effector cells (11, 22). Consequently, a large clone size for Treg is less crucial than for conventional T cells, whereas a broad TCR specificity is more likely to include specificities for the large set of self-Ag and to allow activation and expansion of Treg by cognate self-peptide ligands, which is necessary to rapidly obtain functional regulators. Indeed, the identification of self-cognate Ag recognized by Treg TCR would be a major step in the deciphering of mechanisms underlying the homeostasis of regulatory T cells.

	Acknowledgments

 
We are grateful to A.-M. Balazuc and A. Louise for cell sorting experiments.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by fellowships from Ligue contre le Cancer, Association pour la Recherche contre le Cancer, INSERM, Institut Pasteur, Ministère de la Recherche Française, and Pasteur-Weizmann.

² Address correspondence and reprint requests to Dr. Nicolas Fazilleau at the current address: The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla CA 92037; E-mail address: nicolas{at}scripps.edu or Dr. Manuelle Viguier, Service Dermatologie, Hôpital Saint-Louis, 1 Avenue Claude Vellefaux, 75010 Paris, France; E-mail address: manuelle.viguier{at}sls.aphp.fr

³ Abbreviations used in this paper: Treg, regulatory T cell; HD, healthy donor; MLE, maximum likelihood estimate.

Received for publication January 17, 2007. Accepted for publication July 25, 2007.

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