Co-expression of TNFR2 and CD25 identifies more of the functional CD4⁺FoxP3⁺ regulatory T cells in human peripheral blood

Summary

Previously we found that co-expression of CD25 and TNFR2 identified the most suppressive subset of mouse regulatory T cells (Tregs). Here, we report that human peripheral blood (PB) FoxP3⁺ cells present in CD25^high, CD25^low and even CD25⁻ subsets of CD4 cells expressed high levels of TNFR2. Consequently, TNFR2-expressing CD4⁺CD25⁺ Tregs included all of FoxP3⁺ cells present in CD4⁺CD25^high subset as well as a substantial proportion of FoxP3⁺ cells present in CD4⁺CD25^low subset. CD4⁺CD25⁺TNFR2⁺ cells identified 5-fold greater number of PB CD4 lymphocytes as Tregs than identified by CD4⁺CD25^high cells, and expressed comparable levels of FoxP3⁺ cells as reported CD4⁺CD25⁺CD127^low/− Tregs. Furthermore, this population of cells exhibited the characteristic Treg phenotype, including expression of high levels of CTLA-4, CD45RO, CCR4 and low levels of CD45RA and CD127. Upon TCR stimulation, human PB CD4⁺CD25⁺TNFR2⁺ cells were anergic and markedly inhibited the proliferation and cytokine production of co-cultured T responder cells. In contrast, CD4⁺CD25⁺TNFR2⁻ and CD4⁺CD25⁻ TNFR2⁺ T cells did not show inhibitory activity. Since some non-Tregs express TNFR2, the combination of CD25 and TNFR2 must be used to identify larger population of human Tregs, which may prove to be of diagnostic and therapeutic benefit in cancer and autoimmune diseases.

Introduction

Identification of CD25-expressing CD4 cells in Balb/c mice as regulatory T cells (Tregs) has greatly advanced our understanding of the basic processes that control immune tolerance [1]. These suppressor cells, comprising 5–10% of mouse peripheral CD4⁺ T cells and expressing the X chromosome-encoded forkhead transcription factor, FoxP3, play an important role in preventing immunopathology by suppressing immune responses to autoantigens, however, they also attenuate natural immune responses against tumor antigens [2, 3]. Tregs are a good target for therapeutic manipulation to induce or abrogate immunological tolerance to self and non-self antigens [4, 5].

The quantitative identification and enrichment of viable Treg cells requires reliable surface markers that are selectively expressed on Tregs. Utilization of CD25 to define human Treg is problematic since this IL-2 receptor α chain does not discriminate regulatory from antigen-responsive activated effector T cells (Teffs). Only ~1% of normal human circulating CD4 cells that express the highest levels of CD25, termed CD25^high, reliably exhibit suppressive activity [6, 7] and have high levels of FoxP3 expression [8, 9]. Although the identification of CD25^high as a marker for human Tregs by Baecher-Allan/Hafler and colleagues has greatly advanced in the purification of viable human Tregs [6, 7], it is difficult to accurately quantitate Treg cells based on CD25^high and this criterion may underestimate Treg levels. In addition, the low frequency of CD25^high makes it difficult to isolate sufficient number of Tregs for either in vitro study or for in vivo cellular therapy. Unfortunately, FoxP3 and CTLA-4 expressed by Tregs become detectable only when cells are fixed and permeabilized, and thus can not be used as a biomarker to isolate viable cells. Other reported markers of human Tregs, although helpful in the further characterizing Tregs, also have their limitations when they are used to define human Tregs. For example, HLA-DR expressing Tregs represent an even lower percentage(often <1%) of CD4⁺ T cells [10]. CD27 has been proposed as a surface marker of human Tregs [11], however, the vast majority of PB antigen-activated CD25⁺ effector T cells (Teffs) also express high levels of CD27 [12]. Recently, major progress in the identification of biomarker of Tregs has been made by Liu/Bluestone and Seddiki/Fazekas de St Groth and their colleagues who reported that human Tregs expressed low levels of CD127, the IL-7 receptor α chain, and that the absence of this molecule therefore can be used with CD25 to define human Tregs [9, 13]. Nevertheless, an additional surface marker which positively correlates with FoxP3 expression and immunosuppressive function may improve the identification and isolation of human Tregs.

It has been shown that human thymic CD4⁺CD25⁺ Tregs constitutively express TNFR2, while thymic CD4⁺CD25⁻ cells do not express this receptor [14]. We found that the majority (~80%) of mouse thymic Tregs (CD8⁻ CD4⁺CD25⁺) are also TNFR2-expressing cells [15]. Normally 30~40% of CD4⁺CD25⁺ cells comprised greater than 90% FoxP3⁺ cells in peripheral lymphoid tissues of unstimulated Balb/c mice and C57BL/6 (B6) mice expressed TNFR2, while only <10% of CD4⁺CD25⁻ Teffs were TNFR2⁺ cells and up to 40% of them expressed FoxP3 [15, 16]. Thus, TNFR2 is predominantly expressed on mouse as well as on human Tregs [14, 16]. Furthermore, TNFR2 expression identified the most potent suppressive subset of mouse CD4⁺CD25⁺ Tregs, while CD4⁺CD25⁺TNFR2⁻ T cells in normal C57BL/6 mice have only minimal or no suppressive activity [15]. The findings concerning mouse Tregs could often be extrapolated to human. For example, CD25 as a mouse CD4 Treg marker led to the discovery that CD25^high could define human CD4 Tregs [6]. Recently, it was reported that human CD8⁺CXCR3⁺ cells were actually the counterpart of mouse CD8⁺CD122⁺CXCR3⁺ Tregs [17]. We therefore hypothesized that the expression of TNFR2 might help identify functional human suppressor cells. In this report, we show that human PB CD4⁺CD25⁺TNFR2⁺ T cells also consistently exhibited the phenotypic and functional attributes of Tregs.

Results

Relationship between expression of TNFR2 and FoxP3 on human PB CD4 cells

It was recently reported that TNFR2 expression was highest on human peripheral blood (PB) CD4⁺CD25^high T cells, but could also be detected on CD4⁺CD25^int and on a minor fraction of CD4⁺CD25⁻ T cells [18]. We confirmed this observation and further examined the relationship between TNFR2 and FoxP3 expression in human PB CD4 subsets with different CD25 expression levels, since to date, FoxP3 remains the most specific marker of the Treg lineage [3]. Normal human peripheral lymphocytes contained ~40% of CD4 cells and ~12% of CD25⁺ cells (data not shown). About 1% of the brightest CD25-expressing CD4 cells were defined as CD25^high (or CD25^hi) cells [6]. For convenience sake, all CD25⁺ cells except for the CD25^high were defined as CD25^low (or CD25^lo) cells. Consistent with the report that human PB CD4 cells with high levels of CD25 have repeatedly been shown to be functional Tregs [6], most of the CD25^high cells were FoxP3⁺ and almost all of them (97.4~100%) expressed the highest levels of TNFR2 (Fig 1A). We have found that although usually not considered to be Tregs, human PB CD4⁺CD25^low cells still consisted of about 40% of FoxP3⁺ cells. From 83.3~89.6% of FoxP3⁺ cells in this CD25^low subset were TNFR2⁺ cells while, in contrast, only 36.1~48.4% of FoxP3⁻ presumably effector T cells (Teffs) in this population expressed TNFR2. Furthermore, a small but detectable 3~5% of CD25⁻ cells also expressed FoxP3. 67~83.3% of CD25⁻FoxP3⁺ cells did express TNFR2, while only 16.0~17.9% of CD25⁻FoxP3⁻ cells expressed low levels of TNFR2 per cell (Fig 1A). The high levels of TNFR2 expression on FoxP3⁺ cell, regardless CD25 expression, were consistently observed in this study. The percentage of TNFR2⁺ cells present in CD25^lowFoxP3⁺ and CD25⁻FoxP3⁺ subsets was also markedly higher than in the CD25^lowFoxP3⁻ and CD25⁻FoxP3⁻ subsets, respectively (P<0.01, Fig 1B). Thus TNFR2 and FoxP3 expression in CD4 subsets were directly correlated (Fig 1C). Approximately 85% of PB CD4⁺FoxP3⁺ cells expressed high levels of TNFR2, in contrast, only ~20% of FoxP3⁻ cells expressed low levels of TNFR2. Therefore, both the proportion of cells and the level of expression (MFI) of TNFR2 are highly correlated with FoxP3 expression in human PB CD4 cells.

Figure 1. Human PB FoxP3⁺ Tregs express high levels of TNFR2.

Freshly isolated human PBMCs were stained for CD4, CD25 and TNFR2 and then were fixed and stained intracellularly for FoxP3. CD4 cells were analyzed by FACS, gating on lymphocytes via their forward and side scatter properties and CD4 staining. (A) FoxP3 and TNFR2 expression by CD25^hi, CD25^lo and CD25⁻ subsets of CD4 cells. (B) Percentage of TNFR2-expressing cells in CD4 subsets. (C) FoxP3 and TNFR2 expression by total CD4 cells, CD4⁺CD25^hi cells, CD4⁺CD25^lo cells and CD4⁺CD25⁻ cells. Date shown in (A) and (C) are representative of at least three separate experiments on different donors with similar results. Data shown in (B) are summarized from three different donors (mean ± SEM, N=3). The numbers in the quadrants indicate the percentage of positive cells. The numbers in histograms are mean fluorescence intensity (MFI) and percentage of positive cells (%). Dashed line histogram shows isotype control. Comparison of TNFR2 expression on CD25^loFoxP3⁺ and CD25^loFoxP3⁻ or CD25⁻FoxP3⁻ and CD25⁻FoxP3⁺ cells: ** P<0.01.

Relationship of combination of CD25 and TNFR2 expression in PB CD4 cells to FoxP3 expression

In normal healthy donors, CD25⁺TNFR2⁺ cells represented 5~12% of freshly isolated PB CD4 population (Fig 2A), which is substantially higher than the previously reported 1~2% CD25^high Tregs [6] or <1% HLA-DR⁺ Tregs [10], or 5.8% CD25+CD127^lo/− Tregs [9]. The proportion of human circulating CD4⁺CD25⁺TNFR2⁺ cells is comparable to the reported 5~10% of CD4⁺CD25⁺ Tregs present in mouse peripheral lymphoid tissues [16, 19, 20]. All human CD25^hi cells were TNFR2⁺ and therefore were included in the CD25⁺TNFR2⁺ subpopulation of CD4 cells (Fig 2A).

Figure 2. FoxP3 expression by human PB CD4⁺CD25⁺TNFR2⁺ cells.

Freshly isolated human PBMCs were stained for CD4, CD25 and TNFR2. The cells then were fixed and stained intracellularly for FoxP3. For FACS analysis, the PBMCs were gated on CD4⁺ lymphocytes based on forward and side light scatter and CD4 staining. (A) Expression of CD25 and TNFR2 on PB CD4 cells. Data from three different donors are shown. The numbers in the quadrants indicate the percentage of positive cells. (B) Expression of FoxP3 by CD4⁺CD25^hi cells (upper panel) and CD4⁺CD25⁺TNFR2⁺ cells (lower panel). Data shown are representatives of at least three separate experiments on different donors with similar results. The numbers in the dot plots are the number of gated cells. The numbers in histograms are the percentage of positive cells (%). (C) Expression of FoxP3 by different subsets of PB CD4 cells. The data shown are summarized from eight different donors (mean ± SEM, N=8). Comparison of percentage of FoxP3⁺ cells in CD25⁺TNFR2⁺ subset and CD25⁺TNFR2⁻ subset or CD25⁻TNFR2⁺ subset and CD25⁻TNFR2⁻ subset: ** P<0.001.

Since both CD4⁺CD25^high cells and CD25^lowFoxP3⁺ cells expressed high levels of TNFR2, we hypothesized that co-expression of TNFR2 and CD25 might identify more FoxP3⁺ Tregs. As shown in Figure 2B~C, analysis of multiple donors revealed that greater than 85.3% on average (range: 72.8~94.1%) of CD4⁺CD25⁺TNFR2⁺ cells expressed FoxP3. This is comparable to FoxP3 profile reported for human PB CD4⁺CD25^high cells (79.4~95.5%) [8] and CD4⁺CD25⁺CD127^low/− Tregs (67.4~93.6%) [9]. Consequently, the CD4⁺CD25^high/lowTNFR2⁺ subset included a greater number of cells with FoxP3-expressing Treg phenotype than CD4⁺CD25^high cells. In contrast, CD4⁺CD25⁺TNFR2⁻ cells, uniformly expressed low levels of CD25, only comprised 33.1% on average of FoxP3+ cells (range: 20.0~54.8%), which was markedly lower than the 85.3% FoxP3-expressing cells present the CD25⁺TNFR2⁺ cells (P<0.001). Furthermore, only 18.1% on average of CD25⁻TNFR2⁺ cells were FoxP3⁺ (range: 15.1~22.1%), while ~1% of CD25⁻TNFR2⁻ cells expressed FoxP3 (P<0.001). The intensity of FoxP3 expression on a per cell basis by CD25⁺TNFR2⁺ cells was also higher, than by CD25⁺TNFR2⁻ cells and by CD25⁻ TNFR2⁺ cells (data not shown). Therefore, co-expression of CD25 and TNFR2 is able to identify CD4 Tregs expressing high levels of FoxP3⁺.

Phenotype of human PB CD4⁺CD25⁺TNFR2⁺ cells

Surface expression of antigens by Tregs allows characterization of their phenotype and may also provide insight into their mechanism of action. Mouse CD4⁺CD25⁺TNFR2⁺ T cells represent a subset of Tregs that have a memory/activated phenotype [15]. We therefore investigated whether human PB CD4⁺CD25⁺TNFR2⁺ T cells had similar phenotypic characteristics as their mouse counterparts. This was determined by comparing the HLA-DR, CTLA-4, FoxP3, CD45RO, CD45RA, CCR4 and CCR7 levels of the various subpopulations. As shown in Figure 3, CD4⁺CD25⁺TNFR2⁺ cells expressed the highest level of CD45RO (70.08%), a marker that is associated with proliferative responses to recall antigens, but the lowest level of CD45RA (4.85%), a marker of naïve CD4 cells, similar to the low level expression of CD45RB by mouse TNFR2⁺ Tregs [15], indicative of a memory/activated phenotype. CTLA-4 is expressed predominantly by the Treg compartment of resting mouse CD4 cells and is critical for Treg function [21, 22]. Human CD4⁺CD25⁺TNFR2⁺ cells expressed markedly higher intracellular levels of CTLA-4 (80.24%) than CD4⁺CD25⁺TNFR2⁻ cells (38.45%), CD4⁺CD25⁻TNFR2⁺ cells (42.94%) or CD4⁺CD25⁻TNFR2⁻ cells (9.01%). It has been shown that a CCL22-CCR4 signal was responsible for the trafficking of Tregs to human ovarian tumor [23]. Of the CD4⁺ subsets, CD4⁺CD25⁺TNFR2⁺ cells expressed the highest level of CCR4 (60.06%), while other subsets only contained 12.49~28.39% of CCR4⁺ cells. In contrast, human CD4⁺CD25⁺TNFR2⁺ cells expressed a relatively low level of CCR7 (43.27%), a chemokine receptor responsible for directing traffic of naïve CD4 cells to the lymph nodes, as compared with 60~84% CCR7⁺ cells in the other 3 subsets. As expected, CD4⁺CD25⁺TNFR2⁺ cells were low in expression of CD127[9, 13], indicative of an inverse correlation of TNFR2 and CD127 as markers of Tregs. MHC-DR⁺ Tregs [10] were largely confined to the CD4⁺CD25⁺TNFR2⁺ subset of cells. Thus, in addition to their high level of FoxP3 expression, human CD4⁺CD25⁺TNFR2⁺ cells exhibited the phenotypic characteristics of functional Tregs.

Figure 3. Phenotype of human PB CD4 subsets.

Freshly isolated human PBMCs were stained for CD4, CD25, TNFR2 and additional phenotypic markers. Intracellular expression of CTLA-4 and surface expression of CD45RO, CD45RA, CCR4, CCR7, CD127 and HLA-DR by different subsets of PB CD4 cells, gating on indicated populations were analyzed by FACS. Black filled histogram: antibody staining; grey histogram: isotype control. Numbers in the figures indicate the percentage of gated cells expressing relevant marker. Data shown are representative of at least three separate experiments on different donors with similar results.

Human PB CD4⁺CD25⁺TNFR2⁺ cells are hyporesponsive to TCR stimulation and suppress responder T cells

The in vitro functional hallmarks of Tregs are that they are anergic to TCR stimulation and markedly suppress activation of co-cultured responder T cells. We therefore compared the response to TCR stimulation of CD4⁺CD25⁺TNFR2⁺ and CD4⁺CD25⁻TNFR2⁻ cells. TCR stimulation of CD4⁺CD25⁻TNFR2⁻ Teffs resulted in robust proliferation and INFγ production. In contrast, CD4⁺CD25⁺TNFR2⁺ cells were hyporesponsive, both in term of proliferation (Fig 4A) and INFγ production (Fig 4B), to TCR stimulation. Both CD4⁺CD25⁺TNFR2⁻ cells and CD4⁺CD25⁻TNFR2⁺ cells were more responsive than CD4⁺CD25⁺TNFR2⁺ cells, but less responsive than CD4⁺CD25⁻TNFR2⁻ cells, to TCR stimulation (data not shown).

Figure 4. Comparison of functional capacities of human PB CD4 T cell subsets.

CD4⁺CD25⁺TNFR2⁺ and CD4⁺CD25⁻TNFR2⁻ T cells were FACS-sorted from freshly isolated PBMCs. 2.5×10⁴ cells/well of different CD4 subsets were cultured alone. The cells were stimulated with APCs and anti CD3 Ab for 72 h. Proliferation was determined by [³H] thymidine incorporation assay (A). IFNγ level in the supernatants were determined (B). Data shown are two separate experiments on different donors.

Next, we examined the ability of CD4⁺CD25⁺TNFR2⁺ T cells to suppress the proliferation of co-cultured responder Teffs. As shown in Figure 5A, CD4⁺CD25⁺TNFR2⁺ T cells potently inhibited proliferation of co-cultured Teffs (Fig 5A) in a cell number dependent manner (Fig 5B), while CD4⁺CD25⁺TNFR2⁻ and CD4⁺CD25⁻TNFR2⁺ cells usually did not show inhibitory activity. Since CD4⁺CD25⁺TNFR2⁻ and CD4⁺CD25⁻TNFR2⁺ cells were not anergic and proliferated in response to TCR stimulation (data not shown), the [³H] thymidine incorporation assay might not have revealed their suppressive potential. We therefore performed CFSE dilution assay in which MACS-purified autologous CD4 cells were labeled with CFSE and co-cultured with FACS-purified CD4 subsets. CD4⁺CD25⁺TNFR2⁺ T cells consistently markedly suppressed the replication of responder CD4 cells (Fig 5C) in a dose-dependent manner (Fig 5D). In contrast, the other three CD4 subsets, including CD4⁺CD25⁺TNFR2⁻ and CD4⁺CD25⁻TNFR2⁺ cells, did not show inhibitory activity. The anti-proliferative activity of CD4⁺CD25⁺TNFR2⁺ cells was consistently seen with multiple donors (Fig 5E). Furthermore, CD4⁺CD25⁺TNFR2⁺ T cells almost completely inhibited cytokine (INFγ) production by co-cultured Teffs (Fig 5F). Thus, co-expression of CD25 and TNFR2 on CD4 cells was able to identify functional suppressive human PB CD4 Tregs which were also anergic.

Figure 5. Suppressive activities of human PB CD4 T cell subsets.

CD4⁺CD25⁺TNFR2⁺, CD4⁺CD25⁺TNFR2⁻, CD4⁺CD25⁻TNFR2⁺ and CD4⁺CD25⁻TNFR2⁻ T cells were FACS-sorted from freshly isolated PBMCs. In some experiment MACS-purified CD4 cells were used as responder cells. (A) 2.5×10⁴ cells/well of CD4⁺CD25⁻TNFR2⁻ cells, used as responder cells, were cultured alone or co-cultured with indicated CD4 subsets at ratio of 1:1. Background proliferation (APC alone, cpm: 1888.7) was subtracted. (B) 2.5×10⁴ cells/well of CD4⁺CD25⁻TNFR2⁻ cells were cultured alone or co-cultured with CD4⁺CD25⁺TNFR2⁺ cells at indicated ratios. Proliferation was determined by [³H] thymidine incorporation assay. (C) 5×10⁴ cells/well of CD4 cells were labeled with CFSE, cultured alone or co-cultured with indicated CD4 subsets at 1:1 ratio. (D) 5×10⁴ cells/well of MACS-purified CD4 cells were labeled with CFSE, cultured alone or co-cultured with indicated ratio with FACS-purified CD4⁺CD25⁺TNFR2⁺ cells. After 72 h incubation, dilution of CFSE by responder CD4 cells was determined by FACS. The cultured cells were stimulated with APCs and anti CD3 Ab. (A~D) Representative data from 3 separate experiments on different donors with similar results were shown. (E) Percent inhibition of proliferation of CD4⁺CD25⁻TNFR2⁻ T cells by CD4⁺CD25⁺TNFR2⁻ cells and CD4⁺CD25⁺TNFR2⁺ cells [(CPM (responder cells alone)-CPM (responder cells co-cultured with Treg cells))/CPM (responder cells alone)×100%]. Data shown were summarized from three separate experiments on different donors. (F) MACS-purified CD4 cells and FACS-purified CD4⁺CD25⁺TNFR2⁺ cells were cultured alone or co-cultured at ratio of 1:1. The cells were stimulated with APCs and anti CD3 Ab for 72 h. IFNγ level in the supernatants were determined. Data shown are representative of three separate experiments on different donors with similar results. Data in (E–F) are presented as Mean and SEM.

Discussion

Here we report that the phenotypic and functional characteristics of human CD4⁺CD25⁺TNFR2⁺ Tregs present in normal donor peripheral blood closely resemble mouse CD4⁺CD25⁺TNFR2⁺ Tregs. Virtually all human CD4⁺CD25^highFoxP3⁺ cells plus a substantial proportion of CD4⁺CD25^lowFoxP3⁺ cells expressed TNFR2. Thus, the population of CD4⁺CD25^high/lowTNFR2⁺ cells include 5-fold or more FoxP3⁺ Tregs as compared with the number of Tregs identified by CD25^high. More than 90% of human PB CD4⁺CD25⁺TNFR2⁺ cells were FoxP3⁺, which is comparable to the FoxP3 profile of mouse CD4⁺CD25⁺ Tregs (~90%) [16, 19] and FoxP3 profile of reported human CD4⁺CD25⁺CD127^low/− Tregs (86.6%) [9].

These PB CD4⁺CD25⁺TNFR2⁺ cells co-expressed high levels of FoxP3 and also functioned as Tregs. As revealed in a standard in vitro Treg function assay, CD4⁺CD25⁺TNFR2⁺ cells at a 1:1 ratio to CD4⁺CD25⁻TNFR2⁻ cells reproducibly reduced proliferation by the co-cultured Teffs by greater than 60%. It was previously reported that human PB CD4⁺CD25^high Tregs reduced [³H] thymidine incorporation of co-cultured CD4⁺CD25⁻ responder cells by 69% at day 5 and >98% by day 7 [6]. Thus, the average inhibition of > 60% of CD4⁺CD25⁺TNFR2⁺ cells on day 3 in our study is comparable to the level of inhibition exerted by CD4⁺CD25^high at day 5. We evaluated the suppressive effect on highly proliferative CD4⁺CD25⁻TNFR2⁻ cells were used as responder cells. Other investigators have reported more profound suppression because they used less proliferative CD4 cells or CD4⁺CD25⁻ cells which still contained CD4⁺CD25⁻TNFR2⁺FoxP3⁺ cells as responder cells. Production of cytokines by Teffs is usually more susceptible to inhibition by Tregs than their proliferation. Indeed, at a 1:1 ratio of CD4⁺CD25⁺TNFR2⁺ cells to CD4 responder cells, the production of INFγ by responder cells was almost completely inhibited. Consequently, the subset of human PB CD4⁺CD25⁺TNFR2⁺ cells is profoundly suppressive.

It was recently reported that both human and mouse Tregs were able to shed large amounts of soluble TNFR2 (sTNFR2) upon stimulation with IL-2 and plate-bound anti-CD3, and consequently, the resultant sTNFR2 was proposed to provide a means by which Tregs inhibited the activation of Teffs [18]. However, we did not detect an elevated level of soluble TNFR2 in the supernatant in our in vitro culture of human CD4⁺TNFR2⁺ cells stimulated with APCs and soluble anti CD3 for 72 hrs (data not shown), conditions commonly used to exhibit inhibitory activity of Tregs in vitro [24]. Furthermore, FACS-purified mouse and human CD4⁺CD25⁺TNFR2⁺ cells did not release TNFR2 into the supernatants under same conditions (data not shown). Although CD4⁺CD25⁺TNFR2⁺ cells had potent suppressive capacity, another CD4 subset which also expressed TNFR2, e.g. CD4⁺CD25⁻TNFR2⁺ cells, did not have any suppressive activity, mitigating against an inhibitory role for sTNFR2. Although we have not ruled out a role for sTNFR2 as a basis for suppressive effects of Tregs in vivo, shedding of sTNFR2 can not explain the potent suppression mediated by CD4⁺CD25⁺TNFR2⁺ cells in the in vitro Treg functional assay.

Human PB CD4⁺CD25⁺TNFR2⁺ cells, akin to their mouse counterpart, expressed strikingly high levels of CTLA-4 (Fig 3), as compared with the other CD4 subsets. CTLA-4 has been recently found to play a key role in the Treg suppressive effect in mice [22, 25]. Whether CTLA-4 accounts for the suppressive activity of human PB CD4⁺CD25⁺TNFR2⁺ Tregs will be addressed in a future study.

TNFR2 is one of two receptors for TNF, a pleiotropic cytokine which is a major participant in the initiation and orchestration of inflammation and immunity [26]. Unlike TNFR1 which can mediate cytotoxic effect through its death domain, TNFR2 does not have a death domain and is largely confined to lymphoid cells [26]. Several lines of evidence indicate that TNFR2 acts as a co-stimulator for antigen-driven T cell responses [26–29]. The preferential expression of TNFR2 on mouse and human Tregs suggest that this receptor may mediate the activating effect of TNF on Tregs [16]. Similar to our observation that TNF plus IL-2 selectively promotes the proliferation of mouse FoxP3⁺ Tregs [16], human FoxP3⁺ cells present in the CD4 population were also expanded in response to TNF stimulation in conjunction with IL-2 (data not shown), suggestive of a functional role of TNFR2 in the expansion of human Tregs. Presumably, high levels of TNFR2 expression by human Tregs may mediate a quick response to inflammation followed by a negative feedback attenuation of inflammatory responses that serves to prevent collateral self-tissue damage. Thus, IL-2 is essential for the homeostatic survival and proliferation of Tregs, whereas TNF results in a proliferative expansion of Tregs in an inflammatory environment [15, 16]. A recent report showing that expansion of TNFR2⁺ Tregs with enhanced suppressive activity occurs in malaria patients may reflect this scenario [30].

One of the difficulties in studies of human Tregs is to identify active Tregs present in the subset of CD25^int/low/− CD4 cells with suppressive biological function. For example, it was recently reported that CD45RA⁺FoxP3⁺ “naive Tregs”, which have the capacity to be expanded in vitro into homogenous functional Tregs [31], were CD25^int cells [32]. Our data showed that FoxP3-expressing cells, including those present in CD25⁻ population, expressed high levels of TNFR2 (Fig 1). To date, it is impossible to study the function of CD25⁻FoxP3⁺ cells, since their specific surface markers for viable isolation are not clear. How to exploit TNFR2 expression to identify these CD25⁻FoxP3⁺ cells therefore merit more study.

Taken together, our data demonstrate that the combination of CD4, CD25 and TNFR2 identifies a larger population of functional Tregs in the circulation of normal humans. This population of T cells was practically identical to mouse CD4⁺ Tregs, in manifesting high level expression of FoxP3 and CTLA-4, hypo-responsiveness to TCR cross-linking and potent inhibition of activation of co-cultured T responder cells. Identification of human Tregs with CD4, CD25 and TNFR2 provides over 5-fold better detection and viable enrichment of human Tregs than commonly used CD25^high method for in vitro studies and potentially for in vivo therapy.

Materials and Methods

Cells and reagents

Human peripheral blood enriched in mononuclear cells was obtained from normal donors by leukapheresis (Transfusion Medicine Department, Clinical Center, National Institutes of Health, Bethesda, MD, with an approved human subjects agreement). The blood was centrifuged through Ficoll-Hypaque (Sigma), and PBMCs collected at the interface were washed with PBS and centrifuged through isoosmotic Percoll (Pharmacia, Uppsala, Sweden) gradient. CD4-FITC/-APC, CD25-PE/-PE-Cy5/-APC, CD152 (CTLA-4)-APC, CD45RO-PE-Cy5, CCR4-biotin, CD120b (TNFR2)-biotin, purified CD3 and various fluorochrome conjugated streptavidin were from BD Pharmingen (San Diego, CA). CD45RA-PerCP Cy5.5, CD127-PerCP Cy5.5, HLA DR-PerCP Cy5.5 and FoxP3-APC staining set were from eBioscience (San Diego, CA). CD120b (TNFR2, MR2-1)-PE was from Serotec (Raleigh, NC). CCR7-PE was from R&D systems, Inc. (Minneapolis, MN).

Cell purification, in vitro cell culture and proliferation assay

CD4⁺ cells were purified from freshly isolated human PBMCs with human CD4 microbeads and LS column (Miltenyi Biotec Inc., Auburn, CA). CD4⁺CD25⁺TNFR2⁺, CD4⁺CD25⁺TNFR2⁻, CD4⁺CD25⁻TNFR2⁺ andCD4⁺CD2⁻TNFR2⁻ cells were purified from CD4⁺ cells using Cytomation MoFlo cytometer (Fort Collins, CO), yielding a purity of ~98% for all subsets. Autologous PBMCs were used as APCs by depletion of CD4⁺ cells with anti-human CD4 microbeads (Miltenyi Biotec Inc.). APCs were irradiated with 4,000 R.

For in vitro assays of inhibition of proliferation by Treg, CD4⁺CD2⁻TNFR2⁻ cells (2.5~5×10⁴ cells/well) were seeded in a U-bottom 96-well plate in medium [RPMI 1640 with 10% fetal bovine serum (FBS, Hyclone, Logan, UT) containing 2 mM glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin, 10 mM HEPES]. They were co-cultured with 2×10⁵/well of APCs and 0.5 μg/ml of soluble anti human CD3 Ab. CD4⁺ subsets were added to the wells at a desired ratio to CD4⁺CD25⁻TNFR2⁻ cells. Cells were pulsed with 1 μCi [³H]thymidine (Amersham Pharmacia Biotech, Piscataway, NJ) per well for the last 6 h of a three day culture period. For CFSE dilution assay, CFSE-labelled (2 μM, 8 min at room temperature) responder cells (MACS-purified autologous CD4 cells, 5×10⁴ cells/well) were seeded in a U-bottom 96-well plate together with 2×10⁵ cells/well of APCs and 0.5 μg/ml of anti-CD3 antibody. FACS-purified CD4⁺CD25⁺TNFR2⁺ cells or other CD4 subsets were added to the wells at the desired ratio. After 48~72 h, CFSE dilution was determined with FACS. In some experiments, IFNγ levels in the supernatants of cultures were determined by SearchLight Human Cytokine Array (Pierce Biotechnology, Woburn, MA).

Flow Cytometry

After blocking FcR, cells were incubated with appropriately diluted antibodies. Data were acquired on a FACSort (BD Biosciences, Mountain View, CA) and analysis was conducted using CellQuest software (BD Biosciences).

Statistical analysis

Comparisons of two groups of data were analyzed by two-tailed Student’s t test using Graphpad Prism 4.0.

Abbreviations

PB

peripheral blood

Teffs

effector T cells

Tregs

regulatory T cells