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Keywords:

  • CD4 regulatory cells;
  • regulatory T cells;
  • renal allograft;
  • renal allograft rejection

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. References

We have shown that CD39 and CD73 are coexpressed on the surface of murine CD4+Foxp3+ regulatory T cells (Treg) and generate extracellular adenosine, contributing to Treg immunosuppressive activity. We now describe that CD39, independently of CD73, is expressed by a subset of blood-derived human CD4+CD25+CD127lo Treg, defined by robust expression of Foxp3. A further distinct population of CD4+CD39+ T lymphocytes can be identified, which do not express CD25 and FoxP3 and exhibit the memory effector cellular phenotype. Differential expression of CD25 and CD39 on circulating CD4+ T cells distinguishes between Treg and pathogenic cellular populations that secrete proinflammatory cytokines such as IFNγ and IL-17. These latter cell populations are increased, with a concomitant decrease in the CD4+CD25+CD39+ Tregs, in the peripheral blood of patients with renal allograft rejection. We conclude that the ectonucleotidase CD39 is a useful and dynamic lymphocytes surface marker that can be used to identify different peripheral blood T cell-populations to allow tracking of these in health and disease, as in renal allograft rejection.


Abbreviations: 
Treg

regulatory T cells

Foxp3

forkhead box P3 transcription factor

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. References

CD39 is an ectonucleotidase that is coexpressed with CD73 in the mouse by a subset of CD4+ regulatory T cells (Treg) (1). Extracellular nucleotides, for example, ATP and ADP are hydrolyzed by CD39 to AMP (2); which is subsequently converted to adenosine by CD73 (3). The identification of both CD39 (1) and CD73 (1,4) on murine Treg suggests that adenosine could serve as an important immunomodulatory component of the Treg-suppressive repertoire (1).

In mice, two subpopulations of CD4+CD39+ T cells can be identified. One subset is Foxp3+CD73+ comprising bona fide Treg (1). The other subset, Foxp3CD73, is nonsuppressive and has a memory phenotype (5). This latter group expresses higher levels of mRNA for T-helper (Th) lineage-specific cytokines, typically encompassing all Th1, Th2 and Th17 subtypes. Upon activation, these cells rapidly secrete proinflammatory cytokines. Many murine Treg manifest an unstable phenotype with transient or unstable Foxp3 expression and as such exhibit phenotype plasticity. These ‘exFoxp3’ T cells exhibit an activated memory phenotype and produce inflammatory cytokines such as IFNγ and IL-17A (6).

In humans, the Treg molecular signature is still evolving. The expression of CD39 by human Treg (7) is restricted to a subset of T-regulatory effector memory cells (8) capable of suppressing IL-17 production (9). In some systems, the mechanism by which immunoregulation suppression is exerted is contact dependent (9). Moreover, CD39+ Treg abrogate ATP-dependent effects such as cellular toxicity and maturation of dendritic cells (8).

In contrast to the mouse, Foxp3+CD4+ T cells in human peripheral blood encompass both Treg and non-Treg cells (10). The latter are characterized by the absence of cell surface expression of CD39 (9) and the ability to secrete IFNγ, IL-2 and IL-17, and thereby to include cells with Th17 potential.

We show that within the human CD4+ T cell population the differential expression of CD25 and CD39 can be used to identify four distinct CD4+ T cell populations. CD4+CD25+CD39+ expression identifies a Treg subset while CD4+CD25+CD39 expression denotes a population of T cells with Th17 potential, in accordance with recently published data (8–9). In contrast to the phenotype observed in mice, CD73 is not substantially coexpressed with CD39 in these Treg populations. Moreover, CD39+ expression in the absence of CD25 expression further identifies a memory phenotype, which differentiates pathogenic effector memory cells (11) from regulatory memory cells. Such CD4+CD25CD39+ T cells may represent proinflammatory ‘exFoxp3’ effector memory cells, recently defined in mice (6), which are increased in peripheral blood of patients with antibody-mediated renal allograft rejection.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. References

Human peripheral blood mononuclear cell (PBMC) preparation and Treg isolation

PBMC from controls were prepared by density gradient centrifugation on Ficoll-Paque (GE Healthcare, Uppsala, Sweden). The protocol to obtain volunteer human blood samples was approved by the Beth Israel Deaconess Medical Center Institutional Review Committee. CD4+ T cells were isolated by negative selection using CD4+ no-touch T cell isolation kit (Miltenyi Biotec, Auburn, CA, USA). For some experiments, leukofilters were collected (Blood Donor Center at Children's Hospital, Boston, MA), and CD4+ T cells were isolated using Rosette-sep Human CD4+ T cell isolation kit (Stemcell technologies, Vancouver, Canada) and by density gradient centrifugation on Lymphoprep (Nycomed, Oslo, Norway). Treg were positively selected by staining for CD25 and CD39 using PE or FITC selection kits. Flow cytometry cell sorting (FACS Aria, BD Biosciences, San Jose, CA) was used to obtain highly pure (>98%) Treg (CD4+CD25+CD127low/−).

Antibodies and cytokines

Antibodies used: anti-human FITC-, APC-CD39 (Ancell, Bayport, MN, USA); PE-CD3; PE-Cy5-, PerCP-Cy5.5-, PE-Cy7-CD4; -CD8; -CD19; APC-Cy7-, PE-CD25; PE-IL-17A; Pacific Blue IFNγ; Alexa Fluor 647-CD127 (eBioscience, San Diego, CA); PE-CD127, -CD45RO and -CD73 (BD Pharmingen, Franklin Lakes, NJ); PE-CD44; -CD62; Alexa Fluor 488-, APC-FOXP3 (Biolegend, San Diego, CA). Human recombinant IL-1β, IL-2, IL-23 and TGFβ1 from R&D Systems (Minneapolis, MN).

Cytokine analysis

CD4+ T cells were sorted into CD4+CD25+CD39+/− populations and stimulated with 1 μg/mL PMA and 0.5 μm/mL PHA for 72 h. Supernatant was analyzed by BD Cytometric Bead Array kit (BD Biosciences).

Treg culture methods

FACS-sorted Tregs were activated with anti-CD3-/CD28-coated microbeads (Invitrogen Dynal AS, Oslo, Norway) in a 96-well U-bottom plate. A total of 2 ×105 cells/well were cultured in a 1:1 cell:bead ratio, in a final volume of 250 μL of complete medium (RPMI-1640 containing L-Glutamine (Cellgro, Mediatech Inc, Manassas, VA), 1% nonessential amino acids (BioWhittaker, Walkersville MD), 10% FBS (Gemini Bio-Products, West Sacramento, CA), 50 U/mL penicillin and 50 μg/mL streptomycin [Gemini Bio-Products]). Tregs were cultured in triplicate in the absence or presence of cytokines to induce Th17-polarizing conditions (12).

Intracellular cytokine staining and phenotypic analysis

During the last 4 h of culture, cells were stimulated with PMA (100 ng/mL) and ionomycin (1 μg/mL) in the presence of Brefeldin A (10 μg/mL, all from Sigma-Aldrich, St. Louis, MO). Intracellular cytokine staining was performed according to the manufacturer's instructions using eBioscience Foxp3 staining Kit. The labeled cells were analyzed by flow cytometry on LSRII (BD Biosciences) using FACSDiva (BD) and FlowJo software (Tree Star, Ashland, OR).

End-stage renal failure (ESRF) and transplant patient studies

Patients with ESRF were recruited from the nephrology unit at St. Vincent's Hospital (Melbourne), Australia. Some have received a renal transplant and have been studied in the early postoperative period (postoperative Day 1–6) or at least 5 years posttransplantation with stable allograft function. Written informed consent was obtained. The study was approved by the St. Vincent's Hospital (Melbourne) Human Ethics Committee.

Leukocytes were obtained by Ficoll gradient and CD4+ T cells isolated using MACS microbeads (Miltenyi Biotec). CD4+ T cells were stained with anti-human PE-Cy7-CD4, APC- CD39, APC-Cy7-CD25, PE -CD73, followed by fixation, permeabilization and intracellular staining with Alexa Fluoro 488- Foxp3. Samples were analyzed on SRII Fortessa (BD) using FlowJo software. Statistical analysis was performed using Prism 4.0 software (GraphPad). A one-way ANOVA followed by a Newman–Keuls multiple comparison test was used to determine significance.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. References

CD4+CD25+/−CD39+/− expression identifies four distinct T cell populations

The use of CD4 and CD25 expression to define Treg is inherently flawed as CD25 is also expressed on conventional activated CD4+ T cells. Moreover, resting Treg express lower levels of CD25 expression than activated conventional Treg (10).

The greater proportion of CD25highCD4+ T cells express Foxp3 (Figure 1A). These CD25+ and Foxp3+CD4+ T cell populations contain both CD39+ (1.12 ± 0.85% and 1.56 ± 1.13%, respectively, n = 20) and the very minor CD39 fraction (Figures 1B and C). In addition, CD39 can be shown to be expressed by distinct CD25 and Foxp3 T cell subpopulations (1.37 ± 1.04% and 1.07 ± 0.92%, respectively, n = 20) (Figures 1B and C). The size of these populations are similar to that of CD4+CD25+(Foxp3+)CD39+ T cells and parallel our findings in mice (5).

image

Figure 1. CD4+ T cell populations are defined by differential CD4, CD25 and CD39 expression. (A) Human CD4+ T cells were isolated by Ficoll gradients, stained with anti-human CD25 and intracellularly labeled with Foxp3 then analyzed by FACS. (B, C) Human CD4+ T cells were stained with CD25, Foxp3 and CD39. The additional use of CD39 as a phenotypic marker identifies four distinct CD4+ T cell populations. Mutually exclusive expression of CD73 and CD39 or CD25 by CD4+ T cells. (D, E) PBMCs obtained from healthy controls were stained with anti-human CDH, CD73, CD39 and CD25. Expression of CD73 on CD4+ T cells appeared largely independent of either CD39 or CD25 expression. (F) CD73 was expressed at high levels on CD127high T cells Data denote representative findings for ≥20 independent experiments.

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In mice, CD73 is expressed in tandem with CD39 on the same Treg population (1). However, CD73 is not coexpressed on human CD4+ T cells with either CD39 or CD25 (Figures 1D and E). Indeed, CD4+CD127high T cells express CD73 (Figure 1F) in the resting state. CD73 is upregulated on IFNα-stimulated endothelial cells (13); however, CD73 expression on naïve CD4+CD25 T cells was not altered following stimulation with IFNα (10 U/mL or 100 U/mL) or IFNγ (10 U/mL or 100 U/mL) as determined by flow cytometry at 96 h (data not shown). These data highlight an important difference between the phenotype of murine and human blood Treg ex vivo.

In summary, four distinct T cell populations may be discriminated via differential expression patterns of CD4, CD25 and CD39: CD4+CD25+CD39+; CD4+CD25+CD39; CD4+CD25CD39+ and CD4+CD25CD39 T cells.

CD4+CD25+CD39+ T cells express an activated Treg molecular signature

CD4+CD25+CD39+ T cells comprise approximately 2% of all peripheral blood human CD4+ T cells (data not shown). A majority of these cells (80–100% depending on gating) express Foxp3 (Figure 2A) in abundance (Figure 2B).

image

Figure 2. CD4+CD25+CD39+ T cells have an activated Treg molecular signature. (A) CD4+ T cells were stained with CD39-FITC and CD25-PE and analyzed for intracellular Foxp3 staining (open histogram). (B) The mean fluorescence intensity (MFI) for Foxp3 staining in CD4+CD25+CD39+/- T cells populations is shown (*p < 0.05). (C) PBMCs obtained from peripheral blood were freshly stained without previous in vitro stimulation and stained with anti-human CD25 and anti-human CD127 and labeled with Foxp3. Expression of CD25 is restricted to CD127low population, while expression of Foxp3 extends to the CD127int population. (D) Treg cells identified based on low expression of CD127 and high expression of CD25 were stained with anti-human CD39. CD4+CD25+CD127low T cells contain two distinct populations comprising cells that are either CD39high or CD39low. (E) CD4+CD25+CD127low T cells were stained with anti-human CD39 and labeled with Foxp3. CD39high T cells are largely Foxp3+ while CD39low cells contain an admixture of Foxp3+ and Foxp3 T cells. Data denote representative findings for ≥3 independent experiments.

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To further validate these results, we incorporated staining for CD127 into this study. CD127 (IL-7R) is known to be downregulated on Tregs in human peripheral blood and when used in combination with traditional biomarkers, identifies a highly purified Treg population (14). Although CD25 is restricted to CD127low T cells, Foxp3 expression extends into the CD127int population (Figure 2C). Within the CD4+CD25+CD127low T cell population, two subsets that differentially express CD39 can be identified. In the human PBMCs (n = 20), CD39 is expressed by 70 ± 5% of CD4+CD25+CD127low Treg (Figure 2D). CD4+CD25+CD127lowCD39+ T cells are in general Foxp3+, while CD4+CD25+CD127lowCD39 T cells contain a minor Foxp3 population (Figure 2E).

Lack of CD39 on CD4+CD25+ T cells predicts cells with Th17 potential

Human peripheral blood and lymphoid tissue contain a subpopulation of CD4+Foxp3+ T cells that have the capacity to produce IL-17 (15) and are noted to be CD4+CD25++CD45RA (10). CD4+CD25+CD39 T cells comprise approximately 3% of peripheral CD4+ T cells (not shown) of which 50% express Foxp3 (Figure 2A) albeit at lower mean fluorescence intensity (MFI) compared to CD4+CD25+CD39+ T cells (MFI: 100 vs. 65) (Figure 2B).

CD4+CD25+ T cells isolated from peripheral blood were cultured for 72 h with anti-CD3/CD28-coated microbeads in presence of IL-2, or Th17-promoting conditions (IL-1β, IL-2, IL-23 and TGFβ1). Following culture with IL-2 alone, minimal IL-17A expression was detected by CD4+CD39 T cells, which contained equivalent of Foxp3+ and Foxp3 T cells (Figure 3A).

image

Figure 3. Lack of CD39 expression by CD4+CD25+ T cells with Th17 potential. (A) CD4+CD25+ T cells were stimulated by anti-CD3/CD28 and analyzed for CD39, Foxp3 and IL-17A expression. CD4+CD25+ T cells were cultured in the presence of IL-1β, IL-2, IL-23 and TGFβ1 and then analyzed for CD39, Foxp3 and IL-17A (B); CD39 and IFNγ (C) and IFNγ and IL-17 (D) expression. (E) CD4+CD25+CD39+/− T cells were cultured with PMA for 72 h and the supernatant analyzed for cytokine secretion. CD4+CD25+CD39 T cells (black bars); CD4+CD25+CD39+ T cells (gray bars). Data represent average ± SD (n = 3); *p < 0.05; **p < 0.01. Data are representative of more than three experiments.

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Under Th17-promoting conditions, the number of IL-17A expressing CD4+CD25+CD39 T cells increased 6–8 fold, which was confirmed in three separate experiments. These IL-17A expressing T cells are recruited directly from the CD4+CD25+CD39 T cell compartment and are equally divided between Foxp3+ and Foxp3 T cells (Figure 3B). No IL-17A production is detected in the CD39+ T cells. In the Th17-promoting culture conditions, two-thirds (64%) of IFNγ-producing cells are CD39 (Figure 3C). Under these culture conditions, there are distinct populations of IFNγ and IL-17A-producing cells (Figure 3D).

To examine the impact of CD39 expression on the cytokine profile of CD4+CD25+ T cells, CD4+ T cells were then sorted on the basis of CD25 and CD39 expression into CD4+CD25+CD39+ and CD4+CD25+CD39 T cell populations, cultured for 72 h, and the supernatant was analyzed for various cytokines following stimulation. The putative Treg pool characterized by CD4+CD25+CD39+ expression did not proliferate or produce cytokines. In contrast, CD4+CD25+CD39 T cells proliferated and secreted large amounts of IL-2, IL-4 and IL-5, and lesser amounts of IL-10 and TNFα (Figure 3E). Together, these data highlight the heterogeneous nature of this small T cell population, which encompasses cells with Th1, Th2 and Th17 potential.

CD39 expression denotes memory CD4+ T cell phenotypes and impacts cytokine production

In mice, CD4+CD39+ T cells are subdivided into equivalent proportions of Foxp3+ Treg (1) and Foxp3 effector memory T cells (5). A vast majority of human CD4+CD39+ T cells are CD45RO+ and CD45RA (Figure 4A) and can be divided into CD25+(Foxp3+) and CD25(Foxp3) T cell subsets (Figures 1B and C). Over 80% of CD4+CD25+CD39+ T cells are CD45RO+ denoting a memory Treg phenotype. Moreover, CD45RO is expressed (near universally) by CD4+CD25CD39+ T cells; a pattern consistent with the effector memory T cell phenotype (Figure 4B). A minor fraction (5–33%) of CD4+CD25+CD39 and CD4+CD25CD39 T cells are CD45RO+.

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Figure 4. CD4+ CD39+ T cells are memory T cells. (A) Human CD4+ T cells were stained with anti-human CD39, CD45RO and CD45RA. CD4+CD39+ T cells are CD45RA and CD45RO+. (B) Percentage of CD45RO+ cells within each CD4+ T cell population. Data denote findings for ≥6 independent experiments.

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CD45RO+CD39 T cells were then analyzed for expression of IFNγ and IL-17A by FACS. Under Th17-promoting culture conditions, CD39 expression was consistently induced on 5–7% of CD45RO+ T cells; the majority of which are Foxp3- (Figure 5A). Of importance, CD45RO+ T cells that acquire CD39 expression produce only very low levels of IFNγ and no IL-17A (Figure 5B and C). In contrast, CD45RO+CD39+ memory T cells retain the capacity to produce both IFNγ and IL-17A (Figures 5F and G), which in large part arises from the Foxp3CD39+ T cell population (Figure 5D); the equivalent of murine Foxp3- effector memory T cells (5).

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Figure 5. CD4+CD25CD39+ T cells produce both IFNγ and IL-17A. CD4+CD45RO+CD39 T cells were cultured under Th17 polarizing conditions and analyzed for CD39 and Foxp3 (A), IFNγ (B) and IL-17A (C) expression. CD4+CDRO+CD39+ T cells were cultured under Th17-polarizing conditions and analyzed for CD39 and Foxp3 (D), IFNγ (E) and IL-17A (F) expression. Data are representative of more than three experiments.

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Together these data demonstrate that by utilizing three cell surface markers, CD4+CD25+CD39+ Treg cells can be differentiated from two effector T cell populations (CD4+CD25+CD39 and CD4+CD25+CD39+ T cells), which have the capacity to differentially secrete Th1, Th2 and Th17 proinflammatory cytokines.

CD4+CD25+/−CD39+/- coexpression define four T cell populations in patients with ESRF and following transplantation

The number of peripheral Treg cells as defined by CD4+CD25+ expression is thought to be reduced following renal transplantation (16). The proportion of cells in each CD4+ T cell population based on CD25 and CD39 coexpression was prospectively identified in patients with ESRF (n = 28) awaiting transplantation, patients in the early kidney transplant period (postoperative Day 1–6, n = 10) (Early Tx) and long-term (more than 5 years) renal transplant recipients (Late Tx) with stable allograft function (n = 5) and compared with healthy controls (n = 9). The patterns of expression on CD4+ cells using either CD25/CD39 or Foxp3/CD39 staining patterns were similar in all patient groups (Figure 6A). There was no statistically significant difference between the percentages of cells in these CD4+CD25+/−CD39+/− T cell subsets in controls, patients with ESRF, Early Tx or Late Tx as determined at a time of stable allograft function and in the absence of intercurrent illness.

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Figure 6. Definition of T cell CD4+CD25+/−CD39+/− populations in patients with end-stage renal failure (ESRF) and following renal transplantation. (A) PBMCs were obtained and analyzed by flow cytometry from controls (n = 9), patients with ESRF (n = 28), those in the early transplant period (postoperative Day 1–6) (Early Tx) (n = 10) and patients with stable allograft function at least 5 years posttransplantation (Late Tx) (n = 5). The percentage of CD4+CD25+/−(Foxp3+/−)CD39+/− T cells in each patient group was calculated. There were no significant differences noted between the groups. (B) Representative histological sections of allograft rejection (H&E). (i) Marked interstitial hemorrhage and edema with fibrinoid change in peritubular capillary (arrow). (ii) Severe glomerulitis. (iii) Mild glomerulitis and inflammatory cells in ecstatic peritubular capillaries. (iv) Transmural arteritis with severe intimal arteritis and focal myocyte necrosis (arrows). (C) Peripheral blood CD4+ T cells were obtained from four renal transplant patients during allograft rejection and analyzed for CD4+CD25+/−CD39+/− expression by flow cytometry. The CD4+CD25CD39+ T cell population was increased during allograft rejection compared with both controls and stable late transplant recipients (*p < 0.01). (D) The ratio of CD4+CD25CD39+ (Tmeff): CD4+CD25+CD39+ (mTreg) was determined at the time of allograft rejection. An increase in the Tmeff and concomitant decrease in mTreg was observed.

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In controls, CD4+CD25+CD39+ and CD4+CD25CD39+ T cells are present in equivalent proportions (Figures 1B and C). A similar scenario is evident in late Tx recipients. In patients with ESRF and early following transplantation, around two-thirds of CD4+CD39+ T cells were of the CD25 effector memory T cell phenotype, however; this modest increase did not reach statistical significance compared with controls (data not shown).

With the advent of potent immunosuppressive regimens, the incidence of acute renal allograft rejection is low. The pattern of CD4+CD25+/−CD39+/− T cell expression was followed in four patients presenting with allograft rejection. The Banff staging and scores (17), C4d staining and the presence of donor-specific antibodies (DSA) in these patients are presented in Table 1 with representative histopathological sections depicted in Figure 6B. In the peripheral blood of these patients, the frequencies of CD4+CD25CD39+ T cells were increased (Figure 6C) with concomitant decrease in CD4+CD25+CD39+ T cells. This phenomenon is best appreciated when considering the ratio of CD4+CD25+CD39+ to CD4+CD25CD39+ T cells. In renal allograft rejection, the effector memory pool is increased substantially, when compared to the Treg memory pool, and the magnitude of this increase appeared associated with the severity of rejection. Resolution of rejection biochemically and histologically was associated with more equivalent distributions of CD4+CD39+ T cells (not shown) and in all patients studied, CD73 was not expressed within the peripheral CD4+ T cell population (not shown).

Table 1.  Characteristics of kidney transplant recipients (KTR) presenting with allograft rejection
PatientKTR#1KTR#2KTR#3KTR#4
  1. DSA = donor-specific antibodies.

DiagnosisAcute rejection with interstitial hemorrhage and edema + necrotising capillaritis C4d0Acute antibody-mediated rejection type II with severe glomerulitis + mild intimal arteritis C4d0Chronic active antibody-mediated rejection with mild glomerulitis, PTC mononuclear inflammatory cells and chronic allograft arteriopathy C4d1Acute antibody-mediated rejection type II + acute T cell-mediated rejection with severe glomerulitis and transmural arteritis type III C4d0
Banff acute rejection score17i0t0gl v1 ptc1i0t0 g3 v1 ptc1i1t1 g1 v0 ptc2i2t2g3 v3 ptd1
Banff chronic changes score17ci0ct0 cg0 cv0 ah0 mm0ci0 ct0 cg0 cv1 ah1 mm0ci1 ct1 cg0 cv2 ah1 mm0ci0 ct0 cg0 0c v ah0 mm0
DSANegativeCw5A32; Cw2A29

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. References

In this study, we show that the expression of CD39 on human peripheral CD4+ T cells is intimately associated with regulatory cell (Foxp3+) signatures and the acquisition of a memory (CD45RO+) phenotype. CD39 may allow tracking of dynamic pathogenic effector T cells, recently described in mice as ‘exFoxp3’ cells (6), which are increased in states of inflammation such as in renal transplant rejection and, as recently described, within the joints of patients with childhood arthritis (11). Crucially, the absence of CD39 on CD4+CD25+ T cells discriminates between Treg and a population of cells with Th17 potential and is in agreement with recent data (9).

Tregs are not uniquely defined by CD4+CD25+ expression as this phenotype encompasses many recently activated nonregulatory cells. CD39 together with CD73 have previously been identified as dual surface markers of murine Treg (1,8). Using engineered mice in which GFP expression is driven by Foxp3 gene sequences, we have demonstrated CD39 to be a superior phenotypic marker of Foxp3+ Treg when compared to CD25 in mice (1). Moreover, Foxp3 appears to drive CD39 expression as evidenced by retroviral transduction of CD4+CD25 T cells with Foxp3 (8). Other studies have also demonstrated amplification of the Cd39 gene by Foxp3 (18).

In humans, CD4 and CD25 expression defines a population of T cells with potent suppressive properties (14) that harbors both activated Tregs (CD45RA Foxp3highCD25+++) and cytokine-secreting non-Tregs (CD45RAFoxp3lowCD25++) (10). From a practical perspective, isolating cells on the basis of CD25++ versus CD25+++ (10) carries operation-dependent error and has subjective bias. We propose that staining CD4+CD25+ T cells for CD39 expression better distinguishes both the subsets, where CD4+CD25+CD39+ T cells are activated/memory Tregs and CD4+CD25+CD39 T cells are cytokine-secreting nonsuppressive Tregs. We suggest this approach as an alternative strategy. Indeed, within the highly purified CD4+CD25+CD127low T cell subset, differential levels of expression of CD39 can identify resting from activated Treg, recently defined as CD45RA+Foxp3low(CD25++) and CD45RAFoxp3high(CD25+++), respectively (10).

CD4+CD25+CD39 T cells have been shown to suppress proliferation and IFNγ, but not IL-17A, production (9). We demonstrate that the 50% of these cells express Foxp3 albeit at lower intensity when compared with Treg. We suggest there is considerable overlap between the ‘prototype’ cell surface signature of Treg and those other cells with Th17 potential. Our data further highlight the spectrum of biological activity of CD4+CD25+CD39 T cells. This population not only has IL-17 secreting capacity, but also has the potential to secrete IFNγ, IL-4, IL-5 and IL-10 and encompass differentiating memory CD4+ T cells, Treg, Th1 and Th2 cells (5,15).

Extrapolation of data from murine model systems to human Treg is not always valid. Foxp3, for example, is dynamically regulated in humans in much the same way as CD25. We show that in contrast to the phenotypes observed in mice, CD73 is not consistently coexpressed by isolated human Treg as defined by CD4+CD25+CD39+ under basal conditions within the peripheral circulation of controls or patients and following in vitro activation. Moreover, in the acute inflammatory state of transplant rejection, there was no evidence of specific expression of CD73 on T cell subsets of interest.

In mice, adenosine has been implicated in the mechanism of immune suppression (1) and regulates the function of both the innate and adaptive immune systems through targeting virtually every cell type that is involved in orchestrating an immune response (19). Treg effects may be also due to cell contact-dependent transfer of cyclic adenosine monophosphate(20) the intracellular level of which is increased following signaling through adenosine receptors. Roles for ATP scavenging and metabolic disruptive effects have been also implicated in the antiproliferative T cell responses (8,21).

Given these observations in mice, it is intriguing that CD73 is not coexpressed with CD39 on human Treg. This difference raises a question surrounding the regulatory role of adenosine as a cellular immune suppressive factor in humans. It is possible that the transcellular expression of CD73 is required at the target cell location to generate adenosine from AMP produced by CD39 expressed by Treg. Hence, immune suppressive pathways involving adenosine may be dependent upon paracrine mechanisms requiring the close proximity of other cells expressing CD73 and other ectoenzymes (22). Other pathways involved in the deviation to the Th17 phenotype could involve scavenging of nucleotides by CD39: for example, ATP has recently been shown to drive the differentiation of gastrointestinal-derived Th17 cells (23). Furthermore, tissue-derived adenosine, the downstream product of ATP hydrolysis, acting via the adenosine A2A receptor is an important negative regulator of T cell function (24). Extracellular adenosine inhibits the generation of adaptive effector T cells and drives CD4+ T cells away from Th17 differentiation towards Foxp3+ T cell differentiation. It is also possible that CD73 is restricted to tissue-derived Treg, which has been confirmed in the skin (25) and gastric mucosa (26). These putative pathways could provide a highly regulated and controlled method of immune regulation, which may, however, be disrupted in states of inflammation.

Practically, CD39 can be used for the isolation of functionally active human Treg from the peripheral blood of healthy donors (27). In addition, we show that CD39 in conjunction with CD4 and CD25 clearly distinguishes four distinct T cell populations in controls as well as patients with ESRF and following transplantation. Moreover, these T cell populations are dynamic and can be followed longitudinally. Human cells are more difficult to track with the same precision as mice where genetic tags have developed, however, parallels can be drawn between ‘exFoxp3’ cells (6) and CD4+CD25CD39+ memory effector T cells (5).

In humans, CD4+CD25CD39+ T cells are CD45RO+, Foxp3 and secrete IFNγ and IL-17A. These cells are increased following autoantigen (11) and alloantigen recognition. In the patients presenting with allograft rejection three of the four had antibody-mediated rejection as determined by Banff criteria, C4d staining and/or presence of DSA. Although the histopathological appearances of KTR#1 lacked definitive features of T cell-mediated rejection and were more consistent with antibody-mediated rejection, both DSA and C4d were negative. The detrimental role of antibody in renal allotransplantation is well established and is dominated by endothelial damage secondary to an inflammatory response encompassing complement activation and leukocytes (28). Frequencies of CD4+CD25CD39+ T cells were increased in the peripheral blood of patients with antibody-mediated allograft rejection, the magnitude of which correlated with the severity of rejection as evidenced histologically. Resolution of rejection was associated with a concomitant reduction in the frequencies of CD4+CD25CD39+ T cells to levels observed in nonrejecting transplant recipients.

Frequencies of CD4+CD25+ Treg, decrease following renal transplantation occurring within 2 weeks of the procedure. These changes are significantly impacted on by immunosuppressive agents (16) the effects of which on Treg biology have been well documented (29). Contrary to this other recent report (16), we did not observe a difference in the frequencies of CD4+CD25+/−CD39+/− T cells in healthy controls, patients with ESRF or renal transplant recipients with stable renal allograft function. Further there was no significant difference observed in patients in the early versus late posttransplant phase. The percentage of CD4+CD25+CD39+ T cells in this study was approximately1.5%, which was significantly less than the percentage of CD4+CD25+ T cells observed in the healthy controls and patients with ESRF observed by others. Interestingly, in this other referenced study (16) the percentage of CD4+CD25+ T cells accounted for approximately 2% of all CD4+ T cells following transplantation, which is comparable with values we obtained. This phenomenon may reflect elimination of recently activated cells contaminating the CD4+CD25+ Treg population. Similar data have been obtained in patients with immune activation states such as multiple sclerosis (9,30).

The clinical relevance of Treg tracking in the peripheral blood and the relationships to the equivalent population resident within the graft is not yet established. The differences from peripheral blood sampling to sampling of the graft, the site of allograft rejection and patterns of immune compartmentalization may decrease the sensitivity for detecting changes in lymphocyte populations. As an example, in patients with childhood arthritis only subtle changes are observed in the peripheral blood of patients and controls whereas in contrast within the joint itself, striking changes are evident with a significant increase in the effector memory T cell pool (CD4+CD25CD39+ T cells) (11). Further definition of CD39 expression patterns and leukocyte trafficking within the graft itself may provide useful additional information.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. References

CD39 when used in conjunction with CD4 and CD25 surface lymphocyte staining identifies four distinct and readily identifiable populations in both healthy individuals and those with disease states such as ESRF and following renal transplantation. These T cell populations can be defined and classified as CD4+CD25+CD39+ (activated/memory Tregs); CD4+CD25+CD39 (pro-TH subsets); CD4+CD25CD39+ (memory effector T cells) and CD4+CD25CD39 T cells (naïve/effector T cells). These cellular populations are dynamic and can be tracked with ease in states of inflammation such as in the setting of renal allograft rejection.

Funding sources: This study was funded by KMD: NHMRC, Australia; SCR and TBS: NIH.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Conclusions
  8. References