. 2011 Aug 22;108(35):14602–14607. doi: 10.1073/pnas.1109806108

Quantitative impact of thymic selection on Foxp3⁺ and Foxp3⁻ subsets of self-peptide/MHC class II-specific CD4⁺ T cells

James J Moon ^a,^1,², Pradyot Dash ^b, Thomas H Oguin III ^b, Jennifer L McClaren ^b, H Hamlet Chu ^a,³, Paul G Thomas ^b, Marc K Jenkins ^a

PMCID: PMC3167500 PMID: 21873213

Abstract

It is currently thought that T cells with specificity for self-peptide/MHC (pMHC) ligands are deleted during thymic development, thereby preventing autoimmunity. In the case of CD4⁺ T cells, what is unclear is the extent to which self-peptide/MHC class II (pMHCII)-specific T cells are deleted or become Foxp3⁺ regulatory T cells. We addressed this issue by characterizing a natural polyclonal pMHCII-specific CD4⁺ T-cell population in mice that either lacked or expressed the relevant antigen in a ubiquitous pattern. Mice expressing the antigen contained one-third the number of pMHCII-specific T cells as mice lacking the antigen, and the remaining cells exhibited low TCR avidity. In mice lacking the antigen, the pMHCII-specific T-cell population was dominated by phenotypically naive Foxp3⁻ cells, but also contained a subset of Foxp3⁺ regulatory cells. Both Foxp3⁻ and Foxp3⁺ pMHCII-specific T-cell numbers were reduced in mice expressing the antigen, but the Foxp3⁺ subset was more resistant to changes in number and TCR repertoire. Therefore, thymic selection of self-pMHCII–specific CD4⁺ T cells results in incomplete deletion within the normal polyclonal repertoire, especially among regulatory T cells.

Keywords: tetramer, Treg, tolerance, T-cell receptor, transgenic

According to clonal selection theory, self-reactive lymphocytes are deleted during development, thereby ensuring immunological tolerance to self tissues (1). In the case of T cells, this process occurs in the thymus where developing thymocytes with randomly generated T-cell antigen receptor (TCR) specificities are educated on an array of self-peptide/MHC (pMHC) ligands (2). In the simplest form of this model, thymocytes receiving strong TCR signals are deleted, thereby purging the repertoire of any cells with self-pMHC specificity. The significance of this mechanism of central tolerance has been substantiated by studies demonstrating that impairment of antigen presentation in the thymus can lead to autoimmunity (3, 4). Nevertheless, the clonal deletion rule is challenged by the well-known observation that immunization with certain self-peptides can induce autoimmunity, indicating that at least some responsive self-pMHC–specific T cells routinely exist in the repertoire (5).

Another intriguing exception to the clonal deletion rule is presented by regulatory T (Treg) cells, a suppressive subset of CD4⁺ T cells defined by expression of the Foxp3 transcription factor (6, 7). Whereas some Treg cells are induced from conventional T cells during immune responses (iTreg), most are so-called natural regulatory T (nTreg) cells, which arise directly in the thymus much like naive CD4⁺ T cells (8). Studies have shown that nTreg cells develop as a consequence of the type of high-affinity TCR–self-pMHCII interaction that was thought to cause clonal deletion (9). Moreover, extensive TCR sequencing studies of Treg and non-Treg populations have indicated that, whereas the specificities of these repertoires overlap, Treg cell specificity is biased toward self-pMHCII ligands (10, 11).

These findings lead to the question of why some self-pMHCII–specific CD4⁺ thymocytes undergo clonal deletion, whereas others survive as tolerized Foxp3⁻ cells or differentiate into nTreg cells. This issue has been explored in mice transgenic for both a model antigen and a monoclonal TCR specific for a pMHCII ligand from that antigen. Deletion, anergy, and Treg cell generation have all been described as possible fates for the CD4⁺ T cells in these mice (2, 12, 13). However, the altered timing of TCR expression, the monoclonal nature of the TCRs in question, and the abnormal abundance of T cells expressing these TCRs have raised concerns about the physiological relevance of these findings (14–16).

Here, we improved on this approach by characterizing a polyclonal self-pMHCII–specific CD4⁺ T-cell population within a normal unmanipulated repertoire. Our results indicate that not all self-pMHCII–specific T cells are deleted and that the remaining cells are enriched for nTreg cells and Foxp3⁻ cells with low-affinity TCRs.

Results

Negative Selection to a Ubiquitous Self-Antigen Is Incomplete.

An experimental system was developed on the basis of the model antigen 2W1S (2W), which is a variant of peptide 55–68 of the alpha chain of the mouse MHC class II molecule, I-E^d (17). This peptide is a foreign antigen in C57BL/6 mice (B6), where it is presented on I-A^b molecules and recognized by a large population of naive CD4⁺ T cells (18). To model 2W as a self-antigen, we generated Act-2W transgenic mice, which constitutively and ubiquitously express the 2W peptide embedded within a membrane-bound form of chicken ovalbumin (Fig. S1). pMHCII tetramer-based cell enrichment (18) was used to compare endogenous populations of 2W:I-A^b–specific CD4⁺ peripheral T cells in the spleen and lymph nodes of wild-type (WT) B6 mice, where 2W is a foreign antigen, to those in Act-2W mice, where 2W is a self-antigen. This system thus provided a direct means to study the isolated effect of antigen expression on the development of a single pMHCII-specific T-cell population.

Remarkably, the spleen and lymph nodes of Act-2W mice contained a readily detectable population of 95 2W:I-A^b–specific CD4⁺ T cells per mouse (Fig. 1 A and B). Because 307 2W:I-A^b–specific cells were found in WT controls, this indicated that about 69% of the 2W:I-A^b–specific T cells that naturally develop in B6 mice were deleted when the 2W peptide was present. These numbers correlated well with frequencies measured in the thymus (Fig. 1 A and B). The thymuses of young Act-2W mice contained 52 2W:I-A^b–specific CD4⁺ CD8⁻ single-positive (SP) thymocytes, whereas WT controls contained 177 such cells, reflecting a 71% level of deletion. Thus, ubiquitous expression of 2W antigen resulted in only a partial deletion of the 2W:I-A^b–specific T-cell population, and this occurred predominantly during thymic development.

Tolerant Self-Antigen Specific T Cells Exhibit Low-Avidity Binding.

One possibility was that the 2W:I-A^b–specific T cells in Act-2W mice had low-avidity TCR that did not transduce sufficient signals to induce deletion during thymic development. To assess the TCR binding avidity of these cells, tetramer staining intensity was analyzed, as this has been shown to be directly related to this property (19). Indeed, both peripheral and thymic 2W:I-A^b–specific T cells from Act-2W mice exhibited lower tetramer staining intensity than experiment-matched 2W:I-A^b–specific T cells from WT controls (Fig. 1C). Tetramer titration revealed a 10-fold difference in staining sensitivity between the 2W:I-A^b–specific T cells from Act-2W and WT mice (Fig. 1D). These data indicate that the presence of ubiquitous 2W antigen results in the preferential deletion of high-avidity T cells within the 2W:I-A^b–specific T-cell population.

Act-2W mice were completely healthy and showed no signs of autoimmunity, suggesting that the residual 2W:I-A^b–specific T cells in these animals were tolerant. Indeed, stimulation with direct i.v. injection of 2W antigen or infection with 2W-expressing pathogens did not induce an obvious response from these cells (Fig. 2A). The only stimulus that had a noticeable effect was the s.c. injection of 2W peptide emulsified in complete Freund's adjuvant, which resulted in CD44 up-regulation and a very slight increase in cell number (Fig. 2A). A dose–response showed that the sensitivity of these cells to antigen stimulation was about four orders of magnitude less than that in WT mice (Fig. 2B). Therefore, the peripheral repertoire of T cells with specificity for ubiquitously expressed 2W antigen was comprised of low-avidity cells with a very high threshold of antigen-induced activation.

Fig. 2. — 2W:I-A^b–specific T-cell tolerance in *Act-2W* mice. (A) Representative contour plots of CD4⁺ T cells following 2W:I-A^b tetramer-based enrichment of total lymph node and spleen cells harvested from WT or *Act-2W* mice challenged with the indicated sources of 2W antigen. Numbers indicate total tetramer-positive cells calculated for the entire mouse. Lm-2W, *Listeria monocytogenes*-2W; Sp-2W, *Streptococcus pyogenes*-2W; i.v., intravenous; i.n., intranasal; s.c., subcutaneous. (B) Peptide dose–response for CD44 expression and total numbers of 2W:I-A^b+CD4⁺ T cells 6 d after s.c. injection with CFA. Data depict mean values with SEM from two independent experiments, one mouse per dose.

Clonal Deletion Affects both Foxp3⁺ and Foxp3⁻ Subsets of T Cells.

The preferential deletion of high-avidity T-cell clones within the 2W:I-A^b–specific T-cell population provided a simple explanation for how T-cell tolerance is achieved for this pMHCII ligand. It was also possible that many or all of the residual 2W:I-A^b–specific T cells that survived clonal deletion had become Treg cells. To address this possibility, we assessed expression of the Treg cell marker Foxp3 by either intracellular staining or the use of Foxp3^GFP reporter mice.

As shown in Fig. 3A, 11.8% of the peripheral 2W:I-A^b–specific T cells in Act-2W (or Act-2W × Foxp3^GFP) mice expressed Foxp3, which was only modestly greater than the 7.5% of 2W:I-A^b–specific T cells expressing Foxp3 in WT controls. To investigate this issue more carefully, we enumerated absolute numbers of Foxp3⁺ and Foxp3⁻ 2W:I-A^b–specific T cells in these mice (Fig. 3B). Both subsets of cells were present in significantly lower numbers in Act-2W × Foxp3^GFP mice, indicating that they were both influenced by clonal deletion. There were on average 23 Foxp3⁺ 2W:I-A^b–specific T cells in Foxp3^GFP mice and 12 in Act-2W × Foxp3^GFP mice, indicating a 48% decrease in frequency in the Foxp3⁺ subset. In contrast, there were 316 Foxp3⁻ 2W:I-A^b–specific T cells in Foxp3^GFP mice and 87 in Act-2W × Foxp3^GFP mice, reflecting a 72% decrease in frequency in the Foxp3⁻ subset. Therefore, the ubiquitous expression of 2W antigen influenced the frequency of Foxp3⁻ 2W:I-A^b–specific T cells more than their Foxp3⁺ counterparts.

Effects of Thymic Selection on the 2W:I-A^b–Specific Foxp3⁺ and Foxp3⁻ TCR Repertoires.

We analyzed the 2W:I-A^b–specific TCR repertoire in naive Foxp3^GFP and Act-2W × Foxp3^GFP mice to better understand the qualitative impact of 2W antigen expression. cDNA isolated from single 2W:I-A^b–specific T cells was used to obtain pairs of TCRα and TCRβ gene sequences for each individual cell (20). Almost all of the 127 TCRα and 209 TCRβ gene sequences identified from 2W:I-A^b–specific T cells in Foxp3^GFP mice were unique, confirming earlier reports that the 2W:I-A^b–specific TCR repertoire is very diverse (18).

This high level of diversity would have necessitated an extraordinary amount of TCR sequencing across numerous mice to identify specific sequences preferentially expressed by Foxp3⁺ and Foxp3⁻ 2W:I-A^b–specific T cells. We therefore focused on TCRβ gene segment use patterns. The B6 mouse genome contains 22 unique functional TCR variable region β (TRBV) and 13 unique functional TCR J region β (TRBJ) gene segments, which leads to a total of 22 × 13 = 286 possible unique TRBV/TRBJ combinations (21). We identified 10 TRBV/TRBJ combinations that were overrepresented in the TCR repertoire of 2W:I-A^b–specific T cells in WT mice, occurring in 34.9% of the 2W:I-A^b–specific T-cell population, but only 3.6% of the total CD4⁺ T-cell population (Table 1).

Table 1.

Preferred TRBV/TRBJ combinations of 2W:I-Ab⁺ T cells in WT mice

TRBV/TRBJ combination	Theoretical frequency (%)	Observed frequency in total CD4⁺ T cells (%)	Observed frequency in 2W:IA^b+ T cells (%)
1/2–7	1/286 (0.3)	2/166 (1.2)	7/209 (3.3)
2/2–7	1/286 (0.3)	2/166 (1.2)	16/209 (7.7)
12–1/2–7	1/286 (0.3)	0/166 (0.0)	7/209 (3.3)
13–1/1–2	1/286 (0.3)	1/166 (0.6)	5/209 (2.4)
13–1/2–1	1/286 (0.3)	0/166 (0.0)	5/209 (2.4)
13–1/2–4	1/286 (0.3)	1/166 (0.6)	5/209 (2.4)
13–1/2–7	1/286 (0.3)	0/166 (0.0)	9/209 (4.3)
13–2/1–5	1/286 (0.3)	0/166 (0.0)	6/209 (2.9)
13–2/2–7	1/286 (0.3)	0/166 (0.0)	6/209 (2.9)
16/2–4	1/286 (0.3)	0/166 (0.0)	7/209 (3.3)
Total	10/286 (3.5)	6/166 (3.6)	73/209 (34.9)

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In WT mice, cells with this 2W:I-A^b–specific repertoire signature accounted for 33.9% of 2W:I-A^b–specific Foxp3⁺ cells and 35.3% of 2W:I-A^b–specific Foxp3⁻ cells (Table 2), indicating that the repertoires of these subsets were very similar. In Act-2W mice, cells with the 2W signature comprised 26.9 and 22.0% of 2W:I-A^b–specific Foxp3⁺ and Foxp3⁻ cells, respectively, reflecting losses of cells with the 2W signature in both subsets. However, the 21% decrease in the Foxp3⁺ subset was less than the 38% decrease in the Foxp3⁻ subset, indicating that the presence of 2W antigen resulted in a smaller qualitative change in the Foxp3⁺ TCR repertoire than in the Foxp3⁻ TCR repertoire.

Table 2.

Frequency of preferred TRBV/TRBJ combinations in Foxp3⁺ and Foxp3⁻ subsets

Population	Foxp3⁺ (%)	Foxp3⁻ (%)
Total CD4⁺	3/82 (3.7)	3/84 (3.6)
2W:I-A^b+ from WT	19/56 (33.9)	54/153 (35.3)
2W:I-A^b+ from Act-2W	7/26 (26.9)	18/82 (22.0)

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Foreign-pMHCII–Specific CD4⁺ T-Cell Populations Contain Foxp3⁺ Cells.

Whereas Foxp3⁺ regulatory T cells are known to exist within the repertoire of foreign-pMHCII–specific CD4⁺ T cells, these are generally assumed to be iTreg cells that develop during certain immune responses (8). However, as noted in an earlier study (22), Foxp3⁺ cells were detected in the 2W:I-A^b–specific T-cell population of naive B6 or Foxp3^GFP mice that were never exposed to 2W antigen (Fig. 3A). Most if not all of the Foxp3⁺ 2W:I-A^b–specific T cells in B6 mice expressed the Helios transcription factor (Fig. 4A), a specific marker of nTreg cells (23).

Fig. 4. — Foxp3⁺ T cells in foreign-pMHCII–specific populations. (A) Representative dot plot of Foxp3 and Helios expression in CD4⁺ and 2W:I-A^b+CD4⁺ T cells following enrichment of total lymph node and spleen cells from naive B6 mice. (B) Representative contour plots of CD4⁺ T cells following enrichment of total lymph node and spleen cells from naive *Foxp3^GFP* mice with the indicated tetramers. (C) Scatter plot displaying percentages of Foxp3⁺ cells within each pMHCII-specific T-cell population in individual animals, with mean values indicated. (D) Percentages and absolute numbers of Foxp3⁺ and Foxp3⁻ 2W:I-A^b+ T cells from WT *Tcra*^+/− or *Act-2W Tcra*^+/− mice. Mean values, P values, and limits of detection are indicated.

To determine whether Foxp3⁺ T cells generally exist in other foreign-pMHCII–specific T-cell populations, cell enrichment was performed with I-A^b tetramers containing peptide epitopes from Lymphocytic choriomeningitis virus gp protein (gp 66–77), Listeria monocytogenes listeriolysin O (LLO 190–201), and chicken ovalbumin (OVA 323–339). Due to issues of multiple I-A^b–binding registers within the OVA 323–339 peptide sequence (24), a mixture of two distinct tetramers corresponding to peptides 325–335 (OVA-2) and 327–338 (OVA-3) was used, resulting in the detection of an aggregate population of T cells specific to these two epitopes. As shown in Fig. 4 B and C, each of these foreign-pMHCII–specific T-cell populations contained a subset of cells expressing Foxp3, which ranged in frequency from 8.6 to 12.0%. Thus, thymically derived Foxp3⁺ subsets appear to be a general feature of foreign-pMHCII–specific CD4⁺ T-cell populations.

One possible explanation for the presence of Foxp3⁺ T cells within a foreign-pMHCII–specific population is that these cells possess dual TCRs caused by productive secondary rearrangements of the TCRα gene (Tcra) locus (25). If the specificity of the second TCR is to a self-pMHCII ligand, then agonist selection may drive the cell into a Treg lineage fate. To address this possibility, Foxp3^GFP and Act-2W × Foxp3^GFP mice were crossed with Tcra^−/− mice to create Tcra hemizygous mice (Tcra^+/−) that can only express one αβ TCR type per T cell. The T cells in these mice are thus greatly limited in their ability to bind multiple unrelated pMHCII. As shown in Fig. 4D, the percentage of 2W:I-A^b–specific T cells expressing Foxp3 in these Tcra^+/− mice was 6.0% for WT controls and 12.6% for Act-2W transgenic mice. The absolute numbers of Foxp3⁺ 2W:I-A^b–specific cells in WT Tcra^+/− and Act-2W Tcra^+/− mice were 19 and 11, respectively, and for Foxp3⁻ cells, 310 and 62, respectively. This translated into 42% and 80% reductions in frequency in the respective Foxp3⁺ and Foxp3⁻ subsets. These values were very similar to those in Tcra^+/+ mice (Fig. 3 A and B), indicating that self-pMHCII specificity imparted by a second TCR is not necessary for Treg cell lineage fate in foreign-pMHCII–specific T cells.

Discussion

In this report, we used pMHCII tetramer-based cell enrichment to enumerate and characterize a self-pMHCII–specific T-cell population within a normal unmanipulated polyclonal repertoire. By comparing this population between mice that expressed or lacked the self-antigen, we were able to directly quantify clonal deletion. We found that in the case of a ubiquitously expressed self-antigen, 70% of the T cells with specificity for a pMHCII ligand from that antigen were deleted. This level of deletion was already apparent within the repertoire of CD4⁺ SP thymocytes, which argues against a major role for peripheral mechanisms of steady-state deletion in the shaping of this repertoire.

Our results are largely congruous with previous studies investigating this issue in transgenic mice with limited T-cell repertoires due to expression of a single TCRβ chain with diverse TCRα chains. Bouneaud and colleagues reported that 60–75% of self-pMHCI–specific CD8⁺ T cells were deleted (26). Using a similar strategy, Zehn and Bevan reported a more modest level of deletion (∼24%) (27), but the expression of the self-antigen in this system was restricted to the pancreas, whereas self-antigen expression in the former study was ubiquitous. In our own previous work, we showed that about 90% of thymocytes specific for an abundant IgM-derived pMHCII epitope were deleted (28). Although each of these studies involved a different model self-antigen, they collectively suggest that the extent of clonal deletion in a polyclonal self-pMHC–specific T-cell population is related to the abundance of self-antigen expression in the thymus.

Our finding that 30% of the CD4⁺ T cells specific for a ubiquitous self-pMHCII ligand escape clonal deletion shows that this process is surprisingly incomplete. This percentage may be even higher as tetramer reagents may not detect some T cells expressing TCRs with low affinity for self-pMHCII ligands (29). The simplest explanation would be that these T cells have low avidity TCRs that do not signal strongly enough to induce negative selection, yet can still bind to pMHC tetramers. Our data are consistent with this interpretation, as we observed a lower intensity of 2W:I-A^b tetramer staining in mice expressing 2W antigen. Such a model is also favored by the aforementioned CD8⁺ T-cell studies, which both observed a preferential deletion of T cells with high levels of tetramer binding (26, 27).

It is unclear, however, whether additional mechanisms contribute to the unresponsiveness of the residual 2W:I-A^b–specific T cells in Act-2W mice. Many past studies involving self-pMHC–specific TCR transgenic T cells have documented tolerance through cell intrinsic mechanisms such as down-regulation of TCR sensitivity (30–32) or Treg cell differentiation (9, 33–35). Although the Foxp3⁻ 2W:I-A^b–specific T cells in Act-2W mice have normal amounts of surface TCR and the CD44^low phenotype of naive cells, it is possible that they have unidentified functional defects.

Our study specifically addresses whether self-pMHCII–specific CD4⁺ T cells preferentially become Treg cells. In the case of a CD4⁺ T-cell population specific for a ubiquitously expressed self-pMHCII, we found that very few cells expressed Foxp3. Indeed, expression of the antigen resulted in an overall decrease in the absolute number of Foxp3⁺ pMHCII-specific T cells. This finding is in contrast to previous studies in which transgenic expression of cognate antigen in TCR transgenic mice resulted in the efficient development of self-pMHCII–specific Treg cells (9, 33, 34). It should be noted, however, that these studies reflect the behavior of single TCR transgenic clones under optimal conditions of antigen expression. Therefore, whereas individual clones within a self-pMHCII–specific population may uniformly become Treg cells in the presence of self-antigen, the overall polyclonal self-pMHCII–specific population that we monitored did not behave in this manner. It remains to be seen whether similar results would be obtained in a system involving tissue-restricted self-antigens, where lower levels of thymic self-pMHCII expression or preferential expression in distinct antigen-presenting cell types may be more conducive for Treg cell development in a greater number of clones (36).

The lesser impact of 2W antigen on the frequency of 2W:I-A^b–specific Foxp3⁺ cells than Foxp3⁻ cells is consistent with Foxp3⁺ cells being more resistant to negative selection, as has been reported in TCR transgenic systems (37, 38). However, it is also possible that the presence of 2W antigen may have induced the expression of Foxp3 in T cells that would not have done so otherwise in WT mice. Therefore, the difference in frequency of Foxp3⁺ T cells in WT and Act-2W mice may be the combined effects of clonal deletion through negative selection and increased Treg cell development through agonist selection. Although our TCR repertoire analysis could not identify which specific clones were being deleted by antigen, it showed that the Foxp3⁺ TCR repertoire did not change as much as the Foxp3⁻ TCR repertoire, thereby favoring the simpler former possibility. Either way, the end result would be a higher representation of Foxp3⁺ cells in overtly self-pMHCII–specific than in foreign-pMHCII–specific CD4⁺ T-cell populations.

A surprising part of our study was the discovery of Foxp3⁺ T cells within several naive foreign-pMHCII–specific T-cell populations. Another recent report also identified naive foreign-pMHCII–specific Treg cells (39), supporting our view that nTreg cell subsets are a general feature of both self- and foreign-pMHCII–specific T-cell populations. Moreover, extensive TCR sequencing studies have shown that many TCRs can be found in both Foxp3⁺ and Foxp3⁻ T-cell repertoires (10, 11, 40–42). Thus, whereas TCR signals are a major driving force for Treg development, other contextual signals, perhaps cytokines, are probably also involved (22, 43).

Materials and Methods

Mice and Reagents.

Generation of the Act-2W mouse is described in SI Materials and Methods. B6 and Tcra-deficient mice were purchased from the National Cancer Institute and The Jackson Laboratory, respectively. Foxp3^GFP mice (44) were a gift from Alexander Rudensky (Sloan-Kettering Institute, New York, NY). Act-2W, Foxp3^GFP, and Tcra-deficient mice, and combinations of these strains, were bred in our facilities. Spleen and lymph nodes were taken from 5- to 8-wk-old mice, and thymuses were taken from 5- to 6-wk-old mice. All mice were housed under specific pathogen-free conditions in accordance with University of Minnesota and National Institutes of Health guidelines. All antibodies were purchased from BD Pharmingen, eBioscience, or Life Technologies, unless otherwise indicated. All peptides were ordered from Genscript.

Immunizations.

Mice were injected i.v. with 10⁷ cfu of an attenuated strain of L. monocytogenes transgenically expressing 2W peptide (45), intranasally with 10⁸ cfu of S. pyogenes transgenically expressing 2W peptide, i.v. with 100 μg 2W peptide plus 5 μg lipopolysaccharide (List Biological Laboratories), i.v. with 120 pmol of 2W:I-A^b tetramer, or s.c. with 200 μg 2W peptide in 200 μL of a 1:1 emulsification of PBS and complete Freund's adjuvant (Sigma) distributed evenly between injection sites at the back of the neck, left and right flank, and base of the tail. For the peptide dose–response, s.c. injections were limited to 100-μL volumes distributed evenly between the back of the neck and left flank. For all s.c. immunizations, the spleen and mesenteric lymph nodes were not harvested.

Tetramer Construction.

The generation of peptide:MHCII tetramers is described in SI Materials and Methods.

Tetramer-Based Enrichment and Flow Cytometry.

Tetramer-based enrichment and staining of T cells was performed as described (18), and a detailed protocol is now available (46). Intracellular staining with Foxp3 (eBioscience) or Helios (BioLegend) antibodies was performed with the Foxp3 Staining Buffer kit (eBioscience). Analysis was performed on an LSRII or Fortessa, whereas single cell sorting was performed on a FACSAria II (BD Immunocytometry Systems). Data analysis was performed with FlowJo software (Treestar).

Single Cell TCR Gene Sequencing.

Sequencing of Tcra and Tcrb genes from single sorted T cells was performed as described (20).

Statistical Analysis.

SEM and P values (unpaired two-tailed Student's t test) were calculated using Prism software (GraphPad).

Supplementary Material

Supporting Information

supp_108_35_14602__index.html^{(1KB, html)}

Acknowledgments

We thank Drs. A. Rudensky for Foxp3^GFP mice, S. Way for 2W-expressing L. monocytogenes, and T. Dileepan and P. Cleary for 2W-expressing S. pyogenes. We also thank Drs. J. Kappler, A. Liu, W. Kwok, and T. Cameron for technical expertise and materials related to tetramer construction and J. Walter, R. Speier, A. Schmidt, and M. Preiss for assistance with mice. This work was supported by the National Institutes of Health (F32 AI63793 to J.J.M., R01 AI077714 to P.G.T., R01 AI35296 to M.K.J.), the American Heart Association (fellowship to H.H.C.), the Department of Health and Human Services (HHSN266200700005C to P.G.T.), and the American Syrian Lebanese Associated Charities (P.G.T.).

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1109806108/-/DCSupplemental.

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PERMALINK

Quantitative impact of thymic selection on Foxp3+ and Foxp3− subsets of self-peptide/MHC class II-specific CD4+ T cells

James J Moon

Pradyot Dash

Thomas H Oguin III

Jennifer L McClaren

H Hamlet Chu

Paul G Thomas

Marc K Jenkins

Abstract

Results

Negative Selection to a Ubiquitous Self-Antigen Is Incomplete.

Fig. 1.

Tolerant Self-Antigen Specific T Cells Exhibit Low-Avidity Binding.

Fig. 2.

Clonal Deletion Affects both Foxp3+ and Foxp3− Subsets of T Cells.

Fig. 3.

Effects of Thymic Selection on the 2W:I-Ab–Specific Foxp3+ and Foxp3− TCR Repertoires.