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* Thomas E. Starzl Transplantation Institute and Department of Surgery and
Department of Immunology, University of Pittsburgh, Pittsburgh, PA 15213
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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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
, 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
(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 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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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).
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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.
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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 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 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 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).
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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.
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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 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.
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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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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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.
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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.
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Acknowledgments |
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Disclosures |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Footnotes |
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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.). 
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
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.
Received for publication August 3, 2005. Accepted for publication December 2, 2005.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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 R. H. Thompson, H. Dong, C. M. Lohse, B. C. Leibovich, M. L. Blute, J. C. Cheville, and E. D. Kwon PD-1 Is Expressed by Tumor-Infiltrating Immune Cells and Is Associated with Poor Outcome for Patients with Renal Cell Carcinoma Clin. Cancer Res., March 15, 2007; 13(6): 1757 - 1761. [Abstract] [Full Text] [PDF]
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M. J. Polanczyk, C. Hopke, A. A. Vandenbark, and H. Offner Treg suppressive activity involves estrogen-dependent expression of programmed death-1 (PD-1) Int. Immunol., March 1, 2007; 19(3): 337 - 343. [Abstract] [Full Text] [PDF]
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Y. Y. Lan, Z. Wang, G. Raimondi, W. Wu, B. L. Colvin, A. De Creus, and A. W. Thomson "Alternatively Activated" Dendritic Cells Preferentially Secrete IL-10, Expand Foxp3+CD4+ T Cells, and Induce Long-Term Organ Allograft Survival in Combination with CTLA4-Ig J. Immunol., November 1, 2006; 177(9): 5868 - 5877. [Abstract] [Full Text] [PDF]
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B. Eksteen, A. Miles, S. M. Curbishley, C. Tselepis, A. J. Grant, L. S. K. Walker, and D. H. Adams Epithelial Inflammation Is Associated with CCL28 Production and the Recruitment of Regulatory T Cells Expressing CCR10 J. Immunol., July 1, 2006; 177(1): 593 - 603. [Abstract] [Full Text] [PDF]
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