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The Journal of Immunology, 2006, 176: 2808-2816.
Copyright © 2006 by The American Association of Immunologists

Regulated Compartmentalization of Programmed Cell Death-1 Discriminates CD4+CD25+ Resting Regulatory T Cells from Activated T Cells1

Giorgio Raimondi*, William J. Shufesky*, Daisuke Tokita*, Adrian E. Morelli* and Angus W. Thomson2,*,{dagger}

* Thomas E. Starzl Transplantation Institute and Department of Surgery and {dagger} Department of Immunology, University of Pittsburgh, Pittsburgh, PA 15213


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
More effective discrimination between CD4+CD25+ regulatory T cells (Treg) and activated T cells would significantly improve the current level of purification of Treg and their therapeutic application. We observed that ~90% of Treg (positive for the nuclear transcription factor Forkhead winged helix protein-3 and able to inhibit naive T cell proliferation) isolated from the spleens or lymph nodes of normal mice did not express significant levels of the inhibitory receptor programmed cell death-1 (PD-1) on their surface, but retained PD-1 intracellularly. An identical phenotype was also identified for human CD4+CD25high T cells isolated from peripheral blood of healthy volunteers. By contrast, activated T cells expressed high levels of surface PD-1 that paralleled up-regulation of CD25 during effector cell expansion. This distinction allowed us to isolate CD4+CD25+PD-1– T cells with suppressive activity from mice immunized with mature allogeneic dendritic cells. Although purification was limited to resting Treg because TCR ligation induced up-regulation of surface PD-1, this strategy nevertheless represents a valuable step toward more definitive characterization of Treg and their improved purification for therapeutic assessment.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Regulatory T cells (Treg)3 offer considerable promise for adoptive immunotherapy of autoimmune diseases and allograft rejection (1, 2, 3, 4). Particular interest has focused on so-called naturally arising Treg, a population that appears to represent a unique lineage of T cells within the thymus and that shows potent suppressor activity and comparative abundance (6–12% of the CD4+ T cell population) (3, 4). Recent data demonstrate how these cells can be expanded and used to prevent/cure experimental autoimmune diabetes in transgenic mouse models (5, 6). To be acceptable for clinical use, these cells must exhibit Ag specificity and not compromise the host’s ability to respond to environmental pathogens. Recent publications emphasize the strong potential of Treg to fulfill these criteria (3, 7, 8). In mice, naturally arising Treg are commonly identified as CD4+CD25+ T cells. In animals reared under conventional, specific pathogen-free (SPF) conditions, purified populations isolated using these markers are relatively homogeneous, with suppressor activity. Unfortunately, the isolation and functional characterization of human circulating Treg from patients with perturbations in immune reactivity are hampered by the higher incidence of activated CD4+ T cells that also express CD25 and consequently contaminate CD4+CD25+ Treg preparations.

Many efforts have been made to identify distinctive markers that could be used to discriminate and specifically select Treg. Candidate molecules, such as CTLA-4, glucocorticoid-induced TNFR, and OX40 (CD134), are also expressed by activated CD4+ T cells, whereas others (e.g., CD103 and lymphocyte activation gene-3 (LAG-3)) are expressed by only a limited subset of T cells with regulatory properties (9, 10). Recently, expression of the transcriptional regulator Forkhead winged helix protein-3 (Foxp3) has been associated with T cells with regulatory ability (11, 12, 13, 14). However, its nuclear localization precludes the use of Foxp3 as a tool for Treg isolation. One potentially useful molecule for the identification of Treg is neuropilin-1 (15). Although it appears to be expressed on freshly isolated Treg, but down-regulated by activated T cells (CD25+), the utility of neuropilin-1 expression for functional discrimination of Treg and activated T cells has not been ascertained.

Currently, there is growing interest in elucidation of the role played by coregulatory pathways, in addition to classic CD28-CTLA-4/CD80-CD86 interactions, in the control of immune cell activation. In particular, the receptor molecule programmed cell death-1 (PD-1; CD279), which is up-regulated on activated T cells and other cells, and its ligands B7-H1 (PD-L1; CD274) and B7-DC (PD-L2; CD273), expressed by various hemopoietic cells, may represent a key pathway in the maintenance of peripheral tolerance (16, 17, 18, 19, 20, 21). While investigating the role played by these molecules in the control of Treg function, we have observed that in contrast to activated CD4+ T cells, murine and human Treg express very limited surface PD-1. This observation prompted us to evaluate PD-1 as a discriminatory marker for the separation of Treg from CD4+CD25+ activated T cells.

We show that freshly isolated Treg retain PD-1 in intracellular compartments (in both mouse and human cells), whereas activated CD4+ T cells coexpress surface CD25 and PD-1. This allows ready distinction of the two populations and improves the selective isolation of resting Treg from animals with ongoing immune responses. Moreover, Treg translocate PD-1 to the cell surface when stimulated via the TCR, indicating that purification of CD25+PD-1– T cells renders highly purified resting Treg. This approach may also facilitate investigation of the mechanisms responsible for Treg suppressive activity and their therapeutic potential.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

Eight- to 12-wk-old C57BL/10 (B10; H-2Kb), C3H/HeJ (C3H; H-2Kk), and BALB/c (H-2Kd) mice were purchased from The Jackson Laboratory and maintained in the SPF central animal facility of University of Pittsburgh Medical Center. Experiments were conducted under an institutional animal care and use committee-approved protocol and in accordance with National Institutes of Health-approved guidelines.

Reagents

The following FITC-, PE-, CyChrome (CyC)-, or biotin-conjugated mAbs were used for cytofluorometric analysis: anti-mouse CD4, anti-mouse CD25, anti-mouse CD62L, anti-mouse CD44, anti-mouse CTLA4, anti-human CD4, anti-human CD25 (all from BD Pharmingen), anti-mouse PD-1 (clone J43 (eBioscience) and clone RMP1–30 (Biolegend)), anti-human PD-1, anti-mouse Foxp3 mAb (clone FJK-16s), and anti-human Foxp3 staining set (all from eBioscience). Cells were prepared, stained, and analyzed using a BD Biosciences FACScan flow cytometer and CellQuest or WinMDI software as previously described (22). Foxp3 staining of both murine and human cells was performed following the protocol recommended on the eBioscience web site (<www.ebioscience.com/ebioscience/specs/antibody_72/72-5775.htm>).

T cell purification

CD4+CD25+ T cells were purified from mouse spleens and lymph nodes. Single-cell suspensions were incubated with anti-CD11c, anti-CD11b, anti-CD8{alpha}, anti-B220, anti-Gr-1 (all from BD Pharmingen), and anti-CD49b (eBioscience) biotinylated mAbs. Using streptavidin microbeads and LD depletion columns (Miltenyi Biotec), CD4+ cells were purified by negative selection. This population was then incubated with anti-CD25-PE mAb, and CD4+CD25+ T cells were isolated by positive selection using anti-PE microbeads and MS separation columns (Miltenyi Biotec). Purity was assessed by cytofluorometric analysis and was consistently 90–95%. The remaining cells were used as CD4+ T cells for assay of suppressor function. For isolation of CD4+CD25+PD-1– and CD4+PD-1+ T cells from spleens of immunized animals, the same initial step of negative selection was used to obtain CD4+ T cells. The resulting population was then incubated with biotinylated anti-PD-1 (J43) mAb and the CD4+PD-1+ T cells obtained by positive selection using streptavidin-microbeads and LS separation columns. The CD4+CD25+PD-1– T cell subpopulation was then purified from the remaining cells by positive selection of CD25+ cells as described above. Analyses of human T cells were conducted on PBMC isolated by density gradient centrifugation using Ficoll-Paque Plus (Amersham Biosciences) from 10-ml heparinized blood samples drawn from healthy adult volunteers.

Suppressor function assay

Purified mouse (C3H) CD4+ T cells were CFSE stained using the Vibrant CFDA SE Cell Tracer Kit (Invitrogen Life Technologies) according to the manufacturer’s instructions. CFSE+CD4+ T cells (1.5 x 105) were cultured with 105 autologous, T cell-depleted splenocytes in the presence of soluble anti-CD3{epsilon} (0.6 µg/ml) in 96-well, round-bottom plates using RPMI 1640/10% (v/v) FBS. Graded numbers (8, 4, and 2 x 104) of test cells (CD4+CD25+, CD4+PD-1+, or CD4+CD25+PD-1–) were added to the cultures. CD4+CD25+ T cells were used in all assays as positive controls. After 3 days at 37°C, the cells were harvested, stained for 30 min with anti-CD25-PE (when indicated), and analyzed by flow cytometry.

Intracellular flow staining for PD-1, CTLA4, and Foxp3

Intracellular staining of freshly isolated cells for PD-1 and CTLA4 was performed as previously described (22). Briefly, purified cells were fixed with 4% paraformaldehyde for 20 min, washed, and resuspended in permeabilization buffer (0.1% saponin/1% FBS/PBS) containing biotinylated anti-PD-1 mAb or/and PE-anti-CTLA4. After 30-min incubation, the cells were washed extensively, then incubated with streptavidin-CyC (BD Pharmingen) for 30 min. Permeabilization buffer was used for additional extensive washing. The cells were resuspended finally in PBS and analyzed immediately. Intracellular costaining of PD-1 and Foxp3 (mouse and human cells) was conducted using the Foxp3 staining protocol recommended by eBioscience. Biotinylated anti-PD-1 mAb was added together with anti-Foxp3 mAb, and a second incubation step was performed by addition of streptavidin-CyC.

Dual immunofluorescence staining of cytospins for Foxp3 and PD-1

Cytospins (Shandon cytocentrifuge; 230 x g) of purified murine CD4+CD25+ T cells were fixed in 96% ethanol, blocked with normal goat serum, and incubated overnight (4°C) with the following mAbs: biotinylated anti-PD-1 (clone J43) and anti-Foxp3 (clone MF333F; Axxora). As a second step, slides were incubated with Cy3-streptavidin and Cy2-conjugated anti-rat mAb (Jackson ImmunoResearch Laboratories). Cell nuclei were stained with 4',6'-diamidino-2-phenylinolole (Molecular Probes). The cells were fixed in 2% paraformaldehyde, mounted in glycerol/PBS, and examined with a Zeiss Axiovert 135 microscope equipped with appropriate filters and a cooled CCD camera (CH250; Photometrics).

Murine T cell activation in vitro

Plates (96-well, round bottomed; Corning) were coated overnight (4°C) with 4 µg/ml (PBS) anti-CD3{epsilon} mAb (BD Pharmingen), then washed with PBS. CD4+ T cells (CD25– or CD25+; 1.5 x 105/well) were cultured in RPMI/10% FBS with the addition of anti-CD28 mAb (1 µg/ml final; BD Pharmingen). Alternatively, T cells were activated by coculture (1.5 x 105 cells/well) with 105 autologous, T-depleted, splenocytes and soluble anti-CD3e mAb. Plates were incubated at 37°C for the periods indicated.

In vivo T cell activation

Myeloid dendritic cells (DC) were generated from the bone marrow of B10 mice with rmGM-CSF and IL-4 as previously described (22). During the last day of culture, LPS (100 ng/ml; Escherichia coli O55:B5, Sigma-Aldrich) was added. The resulting mature DC were enriched by gradient centrifugation (500 x g, 20 min at 4°C) over 15% (w/v) Histodenz (Sigma-Aldrich), then washed extensively with PBS. Cells (5 x 106) were injected via the lateral tail vein into BALB/c mice. Three days later, spleens were removed and processed as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Freshly isolated CD4+CD25+ (Foxp3+) T cells with suppressive activity do not express surface PD-1

During studies to establish whether signaling through the inhibitory receptor PD-1 could control the proliferation and function of Treg, CD4+CD25+ T cells were freshly isolated from spleens and lymph nodes of naive C3H mice, and cell surface PD-1 expression was analyzed by flow cytometry (Fig. 1A). The majority (>90%) of CD4+CD25+ cells did not express surface PD-1 or expressed the molecule at very low levels. The same result was obtained with a different mAb clone (RMP1–30).


Figure 1
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  		FIGURE 1. Freshly isolated Treg do not express surface PD-1. A, Flow cytometric analysis of surface PD-1 expression on CD4+CD25+ T cells freshly isolated from lymph nodes and spleen of normal C3H mice. Data are representative of six independent experiments. B, Analysis of intracellular Foxp3 expression on freshly isolated CD4+CD25+ mouse T cells (same as in A). C, CFSE-stained C3H CD4+CD25– T cells were stimulated with soluble anti-CD3{epsilon} mAb in the presence of autologous, T-depleted splenic APC for 72 h, and proliferation was analyzed by the CFSE dilution profile (gray line). CD4+CD25+ T cells were added at the beginning of the culture at a 1:2 Treg:CFSE-stained T cell ratio, and their ability to inhibit proliferation of CFSE-stained cells was determined (black line). Data are representative of three independent experiments. D, The experiment indicated in B was conducted using CD4+CD25+ T cells that were depleted of PD-1+ cells by negative selection with immunomagnetic beads. E, Surface CD25 expression on CFSE-stained T cells stimulated for 72 h, as described in B, in the absence (left) or the presence of CD4+CD25+ T cells (right).

 
The apparent inconsistency with previous reports (6, 23). indicating that Treg express higher PD-1 mRNA levels than naive T cells, prompted us to verify the identity of the isolated CD4+CD25+ T cells as Treg. We first analyzed the intracellular content of Foxp3 (expression of which is restricted, at least in mice, to Treg) (11) by flow cytometry. As shown in Fig. 1B, the purified population had homogeneous expression of Foxp3, confirming the validity of the purification strategy used to purify Treg. We then evaluated the ability of freshly isolated C3H CD4+CD25+ T cells to suppress the proliferation of CFSE-labeled, naive C3H CD4+CD25– T cells in response to soluble anti-CD3{epsilon} mAb and (T cell-depleted) autologous splenic APC. In the absence of added autologous CD4+CD25+ T cells, proliferation of naive T cells that had divided three, four, and five times was clearly evident within 72 h (Fig. 1C). Addition of CD4+CD25+ T cells at a ratio of one Treg to two naive T cells inhibited naive T cell proliferation (Fig. 1C). A proportional increase in replicating T cells was observed with progressive reduction in the Treg:naive T cell ratio (data not shown), with almost complete loss of suppressive activity at a ratio of 1:8. Interestingly, the suppressive function of CD4+CD25+ T cells was not dependent on the presence of the few cells expressing surface PD-1. Thus, removal of PD-1+ cells by magnetic bead sorting did not alter the outcome of the experiment (Fig. 1D).

Additional evidence of the suppressive ability of freshly isolated CD4+CD25+ T cells was obtained by measurement of CD25 expression on CFSE-labeled naive T cells after their stimulation (Fig. 1E). In the absence of added CD4+CD25+ T cells, all dividing populations showed strong expression of surface CD25. Addition of CD4+CD25+ T cells was associated with inhibition of CD25 expression on the few proliferating cells. This effect can be explained by the ability of Treg to inhibit IL-2 secretion by stimulated naive T cells (24) and the consequent reduction in IL-2-induced up-regulation of CD25 normally associated with T cell activation. However, it is also known that CD25 can be up-regulated by an IL-2-independent process (25). Thus, the observed effect could reflect a new suppressive mechanism used by CD4+CD25+ T cells that merits additional investigation. These results clearly indicate that CD4+CD25+PD-1– T cells, which constitute the majority (>90%) of freshly isolated CD4+CD25+ T cells, possess marked suppressive activity and can be identified as naturally arising Treg.

Treg (Foxp3+) retain PD-1 in intracellular compartments

The absence of surface PD-1 expression on >90% freshly isolated CD4+CD25+ T cells was not a strain-specific feature, because it was confirmed in B10, C3H, and BALB/c mice. This observation appears to contradict previous reports describing Treg as cells expressing higher PD-1 mRNA levels than naive T cells (6, 23). The apparent discrepancy could represent another example of a nonlinear relationship between mRNA and protein levels and may, for example, reflect regulated intracellular compartmentalization of PD-1. To address this question, freshly isolated CD4+CD25+ T cells were permeabilized, and the intracellular content of PD-1 was evaluated by flow cytometric analysis (Fig. 2A). Positive staining for intracellular PD-1 was clearly evident. Interestingly, staining of naive CD4+ T cells had another unexpected result. These cells, which do not express any significant surface PD-1 (see Fig. 3A), were positive for intracellular expression of this inhibitory receptor (Fig. 2B). This finding calls for additional investigation of PD-1 expression in relation to the key role played by the molecule in the maintenance of peripheral T cell homeostasis. Confirmation of the intracellular localization of PD-1 in Treg was obtained from cytospins of freshly isolated CD4+CD25+ T cells that were costained for PD-1 and Foxp3 and analyzed by fluorescence microscopy. All CD4+CD25+ cells expressed intracytoplasmic (perinuclear) PD-1 and Foxp3 (Fig. 2C), the latter mainly in the nucleus, as reported previously in Foxp3-GFP Treg (11), confirming the identity of this population as Treg.


Figure 2
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  		FIGURE 2. Freshly isolated Treg retain PD-1 in intracellular compartments similarly to CTLA4. A, Flow cytometric analysis of the intracellular content of PD-1 (open histogram) in permeabilized CD4+CD25+ T cells freshly isolated from lymph nodes and spleen of normal C3H animals (green histogram; isotype control). B, Mouse naive CD4+ T cells obtained after isolation of CD4+CD25+ T cells were analyzed for intracellular expression of PD-1 (red histogram) by flow cytometry (open histogram; isotype control). C, Cytospin preparation of freshly isolated CD4+CD25+PD-1– T cells stained with anti-PD-1 (red) and anti-Foxp3 (green) mAbs. Nuclei were stained with 4',6'-diamidino-2-phenylinolole (blue). Magnification, x100. D, Freshly isolated CD4+CD25+ T cells were analyzed for surface (upper graph) and intracellular (lower graph) CTLA4 expression (open histogram) by flow cytometry (orange and yellow histograms; isotype control). E, Costaining for intracellular PD-1 and CTLA4 in freshly isolated murine CD4+CD25+ T cells. F, Amino acid sequence of murine PD-1, analyzed for the presence of the tyrosine-containing motif YXX0. Two potential interacting motifs (red and green) were identified in the cytoplasmic region (purple) of PD-1.

 

Figure 3
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  		FIGURE 3. Activated mouse CD4+ T cells (initially CD25–) coexpress CD25 and PD-1. A, Kinetic analysis of surface PD-1 expression on CD25+-gated (except for time zero) T cells stimulated with plate-bound anti-CD3{epsilon} and soluble anti-CD28 mAb. Data are representative of two independent experiments. B, CFSE-stained CD4+ T cells were stimulated with plate-bound anti-CD3{epsilon} mAb and soluble anti-CD28. Surface PD-1 expression was analyzed on CD25+-gated T cells 48 and 72 h after stimulation. Data are representative of two independent experiments. C, CFSE-stained CD4+ T cells were stimulated with soluble anti-CD3{epsilon} mAb in the presence of autologous (T-depleted) splenic APC and LPS. Similar to B, surface PD-1 expression (left) was analyzed on CD25-gated T cells at the indicated time and compared with isotype control staining (right).

 
The intracytoplasmic compartmentalization of PD-1 resembles the regulated intracellular localization of the inhibitory receptor CTLA-4 (both proteins belong to the CD28 cosignaling family). It has been documented that although Treg constitutively express CTLA-4, it is retained in intracellular compartments (26, 27). An identical profile was confirmed for CD4+CD25+PD-1– T cells (Fig. 2D). Flow cytometric analysis revealed no CTLA-4 on the surface of these cells, but positive staining within the intracellular compartment. Costaining for intracellular PD-1 and CTLA-4 confirmed their accumulation in Treg (Fig. 2E).

It has been demonstrated that internalization of CTLA-4 (which results in its extremely low surface expression on T cells before their activation) is mediated by an interaction between the adaptor complex AP-2 and a specific cytoplasmic motif of CTLA-4, and that this interaction is regulated by phosphorylation of Tyr165 (28). This prompted us to examine the amino acid sequence of the cytoplasmic region of PD-1 (Fig. 2F) for the presence of the tyrosine-containing motif YXX0, in which a tyrosine residue (Y) is separated from an amino acid with a bulky hydrophobic side chain (0) by two seemingly random amino acids (X), a consensus motif thought to be recognized by adaptor complexes (28). We identified two potential interacting motifs: YEEL, starting at position 225, and YATI, starting at position 248. These loci correspond to parts of the ITIM motif and immunoreceptor tyrosine-based switch motif motifs, respectively, that have been identified in the cytoplasmic portion of PD-1 and that are implicated in the transduction of inhibitory signals that originate from engagement of PD-1 with B7-H1 and B7-DC (16). An analysis of the sequence using NetPhos Server (29) indicated that Tyr225 was the most likely target of phosphorylation.

Activated T cells stably coexpress CD25 and PD-1

Its very low levels of surface expression by Treg indicated that PD-1 might constitute a potential marker to allow separation of activated T cells from Treg (because both populations share surface expression of CD25). We considered that during their activation, T cells express PD-1 (16), and the extent to which this molecule might serve as a discriminatory marker would depend on its stable coexpression together with CD25 on activated T cells. To test this, we measured surface expression of PD-1 on CD25+-gated, activated naive T cells (that were CD25– before TCR triggering) at different time points after their initial stimulation by plate-bound anti-CD3{epsilon} mAb, in the presence of soluble anti-CD28 mAb (Fig. 3A). At 24, 48, 72, and 96 h, CD25+ T cells showed surface expression of PD-1. To obtain fuller characterization of PD-1 expression by activated T cells, we analyzed surface levels of the protein (by flow cytometry) in relation to in vitro cell division. Purified, CFSE-labeled CD4+ naive T cells were stimulated with anti-CD3{epsilon} and anti-CD28 mAbs, as in the previous experiment, and the expression of surface PD-1 was evaluated on CD25+ cells by flow cytometry at 48 and 72 h (Fig. 3B). In agreement with the results shown in Fig. 3A, coexpression of CD25 and PD-1 was stably maintained throughout successive divisions of T cells. In addition, we tested the validity of these findings under activating conditions that better represent the initiation of an immune response. Purified, CFSE-labeled CD4+CD25– naive T cells were stimulated by autologous splenic APC plus soluble anti-CD3{epsilon} mAb and LPS. This stimulation increased the percentage of viable activated T cells (especially at 72 h), as indicated by their forward and side scatter profile during flow cytometric analysis (data not shown). Coexpression of CD25 and PD-1 was confirmed throughout all divisions of T cells (Fig. 3C). Furthermore, the expression of PD-1 by CD25+ T cells increased with the number of cell divisions. These data indicated that in in vitro-activated CD4+CD25– T cells, the surface expression of CD25 is robustly associated with PD-1 expression.

CD4+CD25+PD-1– T cells constitute a population with regulatory activity that can be isolated from spleens of animals with ongoing immune reactions

The foregoing results obtained with activated T cells confirmed the potential of PD-1 as a discriminatory marker, but did not establish its practicality. To test the validity of our hypothesis in vivo, we isolated CD4+CD25+PD-1– T cells with regulatory properties from mice undergoing an immune response. Normal BALB/c mice were injected i.v. with 5 x 106 mature B10 DC (stimulated with LPS; 5 x 106 DC/mouse). Three days after the administration of LPS-stimulated allogeneic DC, spleens, lymph nodes, and blood were obtained from treated and control (untreated) mice, and surface expression of PD-1 on CD4+CD25+ T cells was evaluated (Fig. 4).


Figure 4
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  		FIGURE 4. Injection of mature allostimulatory DC induces two CD25+ T cell populations: PD-1+ and PD-1–. A, DC generated from B10 mouse bone marrow were matured overnight with 100 ng/ml LPS. DC (4–5 x 106) were then injected i.v. into BALB/c mice. Three days later, spleen and lymph nodes cell suspensions were enriched in CD4+ T cells by negative selection. The expression of PD-1 was evaluated on CD25+-gated cells by flow cytometry. Gray histograms are the isotype control. Data are representative of three independent experiments. B, Blood samples were obtained from animals 3 days after injection of mature allostimulatory DC. The expression of PD-1 on CD4+CD25+ circulating T cells (gated) was analyzed by flow cytometry. Data are representative of three independent experiments.

 
In comparison with untreated animals, in which the majority of CD4+CD25+ T cells were PD-1– (Fig. 1A), spleens and lymph nodes of immunized animals contained two subpopulations of CD25+ cells: PD-1– and PD-1+ (Fig. 4A). Interestingly, these two populations were present in approximately similar percentages in the lymph nodes, whereas in the spleen (to which the majority of the i.v.-injected DC home), the PD-1+ subset represented the majority of CD25+ cells. Even in the blood of treated animals there were differences in the incidence of CD4+CD25+ T cells and expression of PD-1 (Fig. 4B). The CD4+CD25+ population constitutes 5% of the circulating CD4+ T cells in normal animals. After injection of mature allogeneic DC, this percentage rose to 15%, and flow analysis of PD-1 expression revealed an increase in the percentage of CD4+CD25+ T cells expressing PD-1 (Fig. 4B). These data confirmed the existence of an alloreactive T cell response and support discrimination between in vivo-activated T cells and Treg based on PD-1 expression on the cell surface. To verify this, spleens were removed 3 days after B10 DC injection, and CD4+PD-1+ and CD4+CD25+PD-1– populations were purified by immunomagnetic bead sorting. The ability of these cells to suppress proliferation of CFSE-labeled, CD4+CD25–, naive T cells (from normal BALB/c mice) after stimulation with autologous splenic APC plus anti-CD3{epsilon} mAb was determined (Fig. 5). As hypothesized, CD4+PD-1+ T cells did not inhibit naive T cell proliferation. By contrast, the presence of CD4+CD25+PD-1– T cells at a ratio of one Treg to two naive T cells significantly suppressed proliferation of the latter population (Fig. 5). It must be noted that compared with the profile obtained using freshly isolated Treg (Fig. 1C), the level of suppression was slightly inferior, as indicated by the presence of a second peak representing cells that had divided once. This limited response might readily be explained by minimal contamination (due to the use of magnetic beads for purification) with PD-1+ cells, representing activated T cells able to secrete IL-2 and, consequently, to support the proliferation of naive T cells, counterbalancing CD4+CD25+PD-1– Treg activities. These results confirmed the feasibility of using PD-1 as a marker to discriminate between activated T cells and Treg.


Figure 5
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  		FIGURE 5. Isolation of CD4+CD25+ T cells with suppressive activity from spleens of mice with ongoing immune reactions. Normal BALB/c mice were injected i.v. with LPS-matured allogeneic DC, and their spleens were removed 3 days later. CD4+PD-1+ and CD4+CD25+PD-1– T cells were purified by magnetic bead isolation, and their suppressive action on CFSE-stained T cell proliferation was examined. The gray line indicates that no test cells were added. Data are representative of two independent experiments with three animals per group.

 
Treg translocate PD-1 to their surface in response to TCR stimulation

The successful isolation of Treg from animals with ongoing alloimmune responses prompted us to consider in more detail the profile of PD-1 expression on freshly isolated CD4+CD25+ T cells (Fig. 1A). It is clear that a minor population (<10% of the cells) expressed significant surface PD-1. We hypothesized that these cells could represent activated T cells contaminating the Treg preparation due to expression of CD25. To verify this, we analyzed the intracellular content of Foxp3 by flow cytometry. Surprisingly, as indicated in Fig. 6A, the majority of these cells were Foxp3+, identifying them as Treg expressing surface PD-1. This result indicates that under certain conditions, Treg can express surface PD-1. An important consideration derives from the observation that expanded Treg express higher PD-1 mRNA levels than freshly isolated Treg (6). This suggests that after activation, Treg may externalize PD-1. To test this hypothesis, we analyzed surface PD-1 expression on freshly isolated Treg 48 and 72 h after in vitro stimulation with anti-CD3{epsilon} and anti-CD28 mAbs (Fig. 6B). Treg up-regulated surface PD-1 after TCR agonism, although with slower kinetics than naive T cells (compare with Fig. 3A). The costaining for surface PD-1 and intracellular Foxp3 72 h after stimulation (Fig. 6C) excluded the possibility that cells expressing PD-1 could represent an expanded population of contaminating non-Treg. It has been shown that Treg stimulated with anti-CD3 exert enhanced suppressive function in vitro (30). This suggests that CD4+CD25+PD-1– T cells, which constitute 90% of CD4+CD25+ T cells, represent resting Treg that patrol tissues, but have not yet been engaged in suppressive activity. The ~10% of freshly isolated CD4+CD25+ T cells that do express PD-1 (Fig. 1A) could therefore mainly represent Treg that are actively inhibiting unwanted immune reactions (whereas activated T cells constitute a very modest fraction, in accordance with the SPF conditions under which the animals are maintained) (31). Additional support for this interpretation comes from analysis of surface PD-1 expression (determined by flow cytometry) on Foxp3+ cells in spleens and lymph nodes of mice injected with mature allogeneic DC (Fig. 6D). In both organs, the presence of two subpopulations (PD-1– and PD-1+) of Treg was clearly evident. Interestingly, a difference in the ratio between these two subpopulations was delineated; in lymph nodes, the majority of Treg were surface PD-1–, whereas in spleen, the dominant population was surface PD-1+ Treg. The association of surface PD-1 expression with Treg activation was substantiated by evaluation of CD62L and CD44 expression on PD-1+ and PD-1– Foxp3+ cells (Fig. 6E) in spleens of injected animals. PD-1– Treg showed a naive phenotype (CD62LhighCD44low), whereas PD-1+ Treg could be divided into two subpopulations according to CD62L expression (high or low). At the same time, the entire population was CD44high, indicating that the cells had been stimulated. These data provide a clear indication of the complex nature of regulation of intracellular vs surface expression of PD-1 in normal T cells and Treg.


Figure 6
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  		FIGURE 6. Activated mouse Treg express surface PD-1. A, Flow cytometric analysis of intracellular Foxp3 expression (gray histogram) in freshly isolated CD4+CD25+ T cells, gated on PD-1+ cells (isotype control; open histogram). B, Freshly isolated CD4+CD25+ T cells were stimulated with plate-bound anti-CD3{epsilon} and soluble anti-CD28 mAbs for the indicated time, and cell surface PD-1 expression was evaluated by flow cytometry. For time 0, refer to Fig. 1A. Open histograms indicate the isotype control. C, Costaining of surface PD-1 and intracellular Foxp3 in CD4+CD25+ T cells stimulated as described in B for 72 h. D, Surface PD-1 profile (open histogram) gated on CD4+Foxp3+ cells obtained from lymph nodes (upper panel) and spleens (lower panel) of BALB/c animals 2 days after injection (i.v.) of B10-derived mature bone marrow-derived DC. E, PD-1– (gray histogram) and PD-1+ (open histogram) Foxp3+ cells from spleens of injected BALB/c animals (as described in D) were analyzed for surface CD44 and CD62L expression by flow cytometry. Data are representative of two independent experiments.

 
Human Treg exhibit a PD-1 profile identical with that of murine Treg

The results obtained suggest the potential of PD-1 as a marker to discriminate between resting Treg and activated T cells and to allow more homogeneous purification of these cells. However, the relevance and possible translational application of these findings is necessarily linked to the demonstration of a similar profile in human Treg. Consequently, we investigated the PD-1 expression profile of peripheral CD4+CD25high T cells (isolated from PBMC) of human healthy volunteers (Fig. 7). As reported previously (1), this population represents the equivalent of murine naturally arising Treg, a fact confirmed by positive intracellular staining for Foxp3 (Fig. 7A). Consistent with the staining of murine Treg, human CD4+CD25high T cells did not exhibit any significant surface PD-1 (Fig. 7B), but showed significant accumulation of intracellular PD-1. In addition to the well-documented expression of PD-1 on the surface of activated human T cells (32, 33), these results provide the basis for additional investigations aimed at delineating PD-1 as an important investigational tool for studies of basic and applied Treg biology.


Figure 7
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  		FIGURE 7. Human Treg express PD-1 but retain the molecule in intracellular compartments similarly to murine Treg. A, PBMC obtained from a healthy human volunteer were analyzed for the presence of CD4+CD25high T cells (upper graph) and the intracellular expression of Foxp3 (open histogram) evaluated on gated population (gray histogram; isotype control). B, The surface (upper graph) and intracellular (lower graph) expression of PD-1 (open histograms) was measured by flow cytometry on the gated CD4+CD25high population (gray histograms; isotype controls).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The results obtained indicate the feasibility of using PD-1 to distinguish activated murine T cells from resting Treg. The identification of a surface marker expressed almost exclusively on activated (non-Treg) T cells could represent a practical advantage compared with molecules expressed specifically on Treg, because the former could, in principle, be safely used during purification of Treg by negative selection. The selective/specific removal of activated T cells expressing surface PD-1 without direct manipulation of Treg minimizes the possibility of altering Treg function. This could represent a potential advantage of PD-1 over CD62L to allow the selection of purer populations of Treg (34, 35). In fact, the latter procedure would require the positive selection of CD4+CD25+CD62L+ by means of specific mAbs. This approach has been successful in murine models, but its translation to humans has yet to be realized; potential undesirable effects could arise from binding of mAb to CD62L (which plays a key role in lymphocyte homing to lymph nodes, where Treg are activated and induced to expand). In addition, it has been shown (36) that, during generation of CD4+CD25+ effector T cells, a consistent fraction (20–40%; including Treg, but the number indicates the presence of a majority of activated T cells) does not down-regulate surface CD62L. This suggests that purification of CD62L+ cells does not allow stringent elimination of activated T cells, whereas up-regulation of PD-1 seems to homogenously characterize the vast majority of effector T cells.

Our unexpected finding of intracellular PD-1 accumulation in Treg raises several questions related to the functional significance of this molecule in Treg homeostasis. It is remarkable that PD-1 behaves very similarly to CTLA4; they are both produced at significant levels, but are retained strictly in intracellular compartments until cell activation. It will be of interest to ascertain the exact localization of PD-1 and CTLA4 in Treg; this could provide an indication of whether similar or different mechanisms are involved in the tight control of their cell surface expression. It is also noteworthy that intracellular accumulation of PD-1 is not restricted to Treg. As indicated in Fig. 2B, naive CD4 T cells express significant amounts of PD-1 that, similarly to Treg, they retain inside the cell (a notable difference in comparison to CTLA4 expression). It would be of considerable interest to investigate whether the third member of this family, B and T lymphocyte Attenuator (16), follows a similar distribution pattern. The intracellular accumulation of PD-1 may represent the necessary storage of an inhibitory molecule that can be promptly exposed (as indicated by its surface expression within 24 h of T cell stimulation; Fig. 3A) and used under specific (as yet unknown) conditions by a healthy immune system to tune its response/control potential autoreactivity (it has to be ascertained whether signals other than TCR stimulation can drive PD-1 to the cell surface).

The purification of Treg from blood of patients with ongoing immune reactions is complicated by the presence of activated T cells that contaminate the isolated CD25+ T cell population. It has been shown that human activated T cells express PD-1 (16, 32, 33). Analysis of PD-1 expression by circulating CD4+CD25+ T cells in mouse blood 3 days after the injection of mature allogeneic DC revealed an increased incidence of PD-1+ cells. In addition, our analysis of peripheral CD4+CD25high T cells from normal human blood that are Foxp3+ confirms that, like mouse Treg, human resting Treg do not express significant amounts of PD-1 on their surface, but accumulate the molecule in intracellular compartments (Fig. 7). Thus, it is conceivable that Treg purification based on the removal of PD-1+ cells to obtain larger numbers and purer populations of Treg from human blood could eventually be applied for therapeutic purposes. According to our in vitro and in vivo results (Fig. 6), it is important to consider that the population resulting from depletion of PD-1+ cells would be deprived of activated Treg. As depicted in the scheme in Fig. 8, our data indicate that unstimulated (resting) Treg do not express surface PD-1. It will therefore be of interest to investigate whether these cells represent Treg directly emigrated from the thymus that maintain PD-1 in intracellular compartments, ready to be externalized in response to stimulation. On the one hand, this represents a potential limitation of the purification approach in terms of its ability to distinguish the entire Treg population (resting plus activated) from activated T cells and indicates that PD-1 is not a lineage marker for Treg. In contrast, it could represent an important advantage, because the more highly purified cells would constitute a homogeneous population of resting Treg for different applications. Indeed, there is considerable interest in defining protocols for in vitro expansion of Treg of defined specificity that could be used in adoptive immunotherapy (6, 37). The ability to start with a population of resting Treg would offer the advantage of having removed any in vivo-activated Treg that could be stimulated to proliferate nonspecifically under in vitro condition, and would allow the expansion of more specific (and effective) Treg populations.


Figure 8
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  		FIGURE 8. Schematic representation of the regulated compartmentalization of PD-1 by Treg and activated T cells.

 
The removal of PD-1+ cells could also benefit the search for a lineage marker that can distinguish Treg (in any functional stage) from other lymphocytes. Analysis of gene expression by a purer population of Treg (from which activated T cells have been removed) will identify markers not evident previously due to the presence of non-Treg. Moreover, the potential to distinguish resting from activated Treg will enable investigations to further elucidate the biology of these cells. With the separation of these two subpopulations, it will be possible to compare their gene expression profiles and identify candidate molecules involved in the multiple processes responsible for their regulatory activity. In addition, the recently reported selective expression of LAG-3 by stimulated CD4+CD25+ Treg (that do not express this molecule when quiescent) (17, 18) should be considered. Selection based on combinations of PD-1 and LAG-3 expression (on the CD25+ population) should result in successful isolation of the following populations: resting Treg (PD-1–), activated Treg (PD-1+LAG-3+), and activated T cells (PD-1+LAG-3–; Fig. 7).

Our data, obtained after in vivo immune activation, indicate that surface PD-1+ Treg are activated cells. This calls attention to freshly isolated CD4+CD25+ T cells, of which <10% are surface PD-1+ (Fig. 1A). This small population may represent the subset of Treg that, at the time of that isolation, are involved in control of unwanted reactions. It would be of interest to ascertain the influence of PD-1+ cell depletion on the efficacy of vaccines for specific types of tumors that are associated with active control of the antitumor response by Treg.

Posttranslational controls can define markers that cannot be identified by analysis of the gene expression profile of specific cell populations. The regulated compartmentalization of PD-1 represents a straightforward example. Additional investigation will be necessary to better clarify the role played by this tight regulation in relation to the demonstrated important contribution of PD-1 to the control of peripheral tolerance. In addition, regulated cell surface expression of PD-1 by Treg may contribute to the development of successful strategies to exploit the potential of selected populations of Treg to control pathological immune responses.


   Acknowledgments
 
We thank Dr. Martin Schmidt for helpful advice on PD-1 sequence analysis, Dr. Penny Morel for valued discussion on manuscript improvement, Dr. Mike Turner (laboratory of Dr. Olja Finn) for helpful feedback on Foxp3 staining, and Miriam Meade for excellent administrative support.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
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 National Institutes of Health Grants R01DK49745, R01AI41011, and R01AI60994 (to A.W.T.) and R01HL077545, R01HL075512, R21HL69725, and R21AI55027 (to A.E.M.). Back

2 Address correspondence and reprint requests to Dr. Angus W. Thomson, Thomas E. Starzl Transplantation Institute and Departments of Surgery and Immunology, University of Pittsburgh, 200 Lothrop Street, W1540, Pittsburgh, PA 15213. E-mail address: thomsonaw{at}upmc.edu Back

3 Abbreviations used in this paper: Treg, naturally arising regulatory T cell; CyC, CyChrome; DC, dendritic cell; Foxp3, Forkhead winged helix protein-3; LAG-3, lymphocyte activation gene-3; PD-1, programmed cell death-1; SPF, specific pathogen free. Back

Received for publication August 3, 2005. Accepted for publication December 2, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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