Human T cells express CD25 and Foxp3 upon activation and exhibit effector/memory phenotypes without any regulatory/suppressor function
© Kmieciak et al; licensee BioMed Central Ltd. 2009
Received: 22 July 2009
Accepted: 22 October 2009
Published: 22 October 2009
Foxp3 has been suggested to be a standard marker for murine Tregs whereas its role as marker for human Tregs is controversial. While some reports have shown that human Foxp3+ T cells had no regulatory function others have shown their role in the inhibition of T cell proliferation.
T cell activation was performed by means of brayostatin-1/ionomycin (B/I), mixed lymphocyte reaction (MLR), and CD3/CD28 activation. T cell proliferation was performed using BrdU and CFSE staining. Flow cytometry was performed to determine Foxp3 expression, cell proliferation, viabilities and phenotype analyses of T cells.
Both CD4+ and CD8+ T cells expressed Foxp3 upon activation in vitro. Expression of Foxp3 remained more stable in CD4+CD25+ T cells compared to that in CD8+CD25+ T cells. The CD4+CD25+Foxp3+ T cells expressed CD44 and CD62L, showing their effector and memory phenotypes. Both FoxP3- responder T cells and CD4+FoxP3+ T cells underwent proliferation upon CD3/CD28 activation.
Expression of Foxp3 does not necessarily convey regulatory function in human CD4+CD25+ T cells. Increased FoxP3 on CD44+ effector and CD44+CD62L+ memory T cells upon stimulation suggest the activation-induced regulation of FoxP3 expression.
In mice, scurfy mutation in forkhead/winged helix transcription factor gene Foxp3 causes autoimmune lesions including massive lymphoproliferation, diabetes, exfoliative dermatitis, thyroiditis and enteropathy. Such autoimmunity can be cured by a transgene encoding a wild-type Foxp3 allele . The expression of Foxp3 in CD4+CD25+ T cells in wild-type mice and the diminished numbers of these T cells in scurfy and Foxp3-knockout (Foxp3-) mice suggested a role for Foxp3 in the development of regulatory T cells (Tregs) . In addition, Foxp3 has been shown to be a specific marker for murine CD4+ Tregs because activation of non-T regs did not induce Foxp3 expression . Ectopic expression of Foxp3 was shown to be sufficient to activate a program of suppressor function in peripheral murine CD4+ T cells .
In humans, the gene encoding Foxp3 was discovered during efforts to understand the genetic basis for a rare X-linked fatal autoimmune disease known as IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome [3, 4]. However, the role of Foxp3 as a key marker for Tregs in humans remains controversial. Unlike mice, activation of human CD4+ T cells by T-cell receptor (TcR) stimulation resulted in the expression of Foxp3 [5–12]. Most of these studies showed that induction of Foxp3, even in the presence of TGF-β, did not correlate with suppressive function of CD4+ T cells [6, 10–12]. Although it was suggested that lack of suppression during the activation-induced expression of Foxp3 in human CD4+ T cells was because of transient expression of Foxp3, the observation still argues against a role for Foxp3 as key regulator of suppression in human CD4+ T cells upon expression. Regardless of the status of Foxp3, many studies considered CD4+CD25high as Tregs in humans without being able to show their regulatory functions in vivo [13–15]. Most recently, it was reported that maternal alloantigens promoted development of Tregs in the human fetus that could suppress fetal antimaternal immunity. The authors considered CD4+CD25+Foxp3+ T cells as Tregs because of their partial suppressive function in a mixed lymphocyte reaction (MLR) in vitro . These controversial reports prompted us to determine whether induction of Foxp3 expression in human T cells during activation and during MLR may confer regulatory functions. Our studies showed that activation-induced expression of Foxp3 was transient in CD8+CD25+ T cells but it was more stable in CD4+CD25+ T cells. These Foxp3+ T cells were mainly of effector and memory phenotypes.
PBMC were collected from two healthy donors, and duplicate experiments were performed.
Three-color staining and FACS analyses were performed as previously described by our group . Extracellular staining were performed using anti-human antibodies from Biolegend: PE- and FITC-CD25 (clone BC96), PE- and FITC-CD44 (clone IM7), FITC-CD62L (clone DREG-56), PE/Cy5-CD4 (clone OKT4) and PE/Cy5-CD8 (clone RPA-T8). Appropriate isotype control antibodies were used to exclude nonspecific binding. Foxp3 intracellular staining was done with PE anti-human Foxp3 Flow Kit (Biolegend, clone 206D) according to the manufacturer's protocol. Apoptosis was determined by staining of cells with Annexin V (BD Pharmingen).
FITC BrdU Flow Kit (BD Pharmingen) was used in proliferation assays. T cells were also labeled with CFSE by incubation at 5 × 107 cells/mL in 5 μM CFSE/HBSS for 5 min at room temperature. Cells were then added with an equal volume of FBS, followed by three washes in FBS-containing HBSS.
Mixed lymphocyte reaction (MLR)
Blood samples were diluted two-fold with PBS and layered onto Ficoll-Hypaque. Each tube was centrifuged at 400 g for 30 min and the lymphocytes at the interface were collected. These cells were washed once with RPMI 1640 medium containing 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine. They were then resuspended at l07 cells/ml in the same medium containing 10% heat inactivated FBS. Allogeneic stimulating cells were irradiated in a cesium irradiator to a total dose of 5,000 rad, to abolish their capacity to proliferate. Cultures were set up in flat-bottomed 24-well plates and 3 × 106 responder cells were mixed with 2 × 106 irradiated stimulators in 2 mL. Cultures, set up in triplicates, were incubated for 8 days at 37°C. Control cells cultured with medium containing low dose IL-2 (20 U/mL) in order to maintain T cell viability during a 3-day culture. No IL-2 or anti-CD3 Ab was used in MLR samples. Some cultures were pulsed with 10 μM BrdU (BD Pharmingen).
Statistical comparisons between groups were made using the Student t test with P < 0.0.5 being statistically significant.
Results and discussion
Activation of T cells induces expression of CD25 and Foxp3 associated with effector and memory phenotype differentiation
Activation-induced FoxP3 expression in CD4+ T cells fails to convey regulatory function in vitro
Allogeneic activation of T cells during MLR induces Foxp3 expression in CD4+CD25+ T cells associated with effector/memory phenotype
In conclusion, the present study shows that Foxp3 expression is not a reliable marker for human Tregs. T cell activation, CD4+ T cells in particular, is associated with the expression of Foxp3 in effector/memory T cells without detectable regulatory function when present at physiologically relevant ratios.
peripheral blood mononuclear cells
activation induced cell death
mixed lymphocyte reaction
- T regs:
regulatory T cells.
This work was supported by NIH R01 CA104757 grant (M. H. Manjili) and Massey Cancer Center Pilot Project Program, 646564. We gratefully acknowledge the support of VCU Massey Cancer Centre and the Commonwealth Foundation for Cancer Research.
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