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Comparative Study
. 2009 Feb 1;182(3):1469-80.
doi: 10.4049/jimmunol.182.3.1469.

Human circulating CD4+CD25highFoxp3+ regulatory T cells kill autologous CD8+ but not CD4+ responder cells by Fas-mediated apoptosis

Laura Strauss  1 , Christoph Bergmann, Theresa L Whiteside
Affiliations
Comparative Study

Human circulating CD4+CD25highFoxp3+ regulatory T cells kill autologous CD8+ but not CD4+ responder cells by Fas-mediated apoptosis

Laura Strauss et al. J Immunol. .

Abstract

Mechanisms utilized by human regulatory T cells (Treg) for elimination of effector cells may vary. We investigated the possibility that the mechanism of Treg suppression depends on Fas/FasL-mediated apoptosis of responder cells (RC). CD4(+)CD25(high)Foxp3(+) Treg and autologous CD4(+)CD25(-) and CD8(+)CD25(-) subsets of RC were isolated from blood of 25 cancer patients and 15 normal controls and cocultured in the presence of OKT3 and IL-2 (150 or 1000 IU/ml). Suppression of RC proliferation was measured in CFSE assays. RC and Treg apoptosis was monitored by 7-aminoactinomycin D staining in flow-based cytotoxicity assays. Treg from all subjects expressed CD95(+), but only Treg from cancer patients expressed CD95L. These Treg, when activated via TCR plus IL-2, up-regulated CD95 and CD95L expression (p < 0.001) and suppressed CD8(+) RC proliferation (p < 0.001) by inducing Fas-mediated apoptosis. However, Treg cocultured with CD4(+) RC suppressed proliferation independently of Fas/FasL. In cocultures, Treg were found to be resistant to apoptosis in the presence of 1000 IU/ml IL-2, but at lower IL-2 concentrations (150 IU/ml) they became susceptible to RC-induced death. Thus, Treg and RC can reciprocally regulate Treg survival, depending on IL-2 concentrations present in cocultures. This divergent IL-2-dependent resistance or sensitivity of Treg and RC to apoptosis is amplified in patients with cancer.

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Figures

FIGURE 1
FIGURE 1
FLOCA simultaneously measures induction of apoptosis and suppression of RC proliferation by CD4+CD25highFoxp3+ Treg. FLOCA was used to simultaneously measure proliferation of CD8+ CFSE-labeled autologous RC and their apoptosis. Treg were CFSE−. CD8+ RC alone or RC plus Treg were stimulated with OKT3 and cocultured in the presence of 150 IU/ml IL-2 for 5 days. At harvest, cells were stained with 7-AAD, which stains dead cells and is excluded by live cells, and examined by flow cytometry. FLOCA discriminates CFSE-labeled 7-AAD+ and 7-AAD− RC from unlabeled 7-AAD+ and 7-AAD− Treg after coculture. A, Treg isolated from PBMC of a NC induced low levels of inhibition and little apoptosis in RC. The data are from 1 experiment of 15 performed with T lymphocytes of NC. B, A representative experiment of 10 performed with T cells of HNSCC patients. Treg isolated from PBMC of a HNSCC patient induced strong inhibition of RC proliferation (89%) and death in 80% of RC. In this and all other figures, the Treg/RC ratio was 1:1 in the cocultures. The percentages of proliferation inhibition and of 7-AAD+ cells are indicated in respective panels.
FIGURE 2
FIGURE 2
Human Treg suppress proliferation but do not mediate apoptosis in CD4+ RC. Proliferation and death (% 7-AAD+) of CD4+ RC alone or CD4+ RC in cocultures with Treg isolated from the peripheral blood of a NC and a HNSCC patient were measured in FLOCA. Note that in the coculture with the patient’s cells nearly all CD4+CSFE− Treg are 7-AAD+ following incubation with CD4+ RC in the presence of OKT3 and 150 IU/ml IL-2. No death is evident in CD4+ RC, although their proliferation is suppressed. In contrast, cocultures set up with NC cells show no Treg apoptosis and little suppression of RC proliferation. Results are representative experiments of 10 performed with cells isolated from PBMC of HNSCC patients and 10 performed with cells isolated from PBMC of NC.
FIGURE 3
FIGURE 3
The role of IL-2 in death of Treg in autologous Treg/CD4+ RC cocultures. A, Percentages of 7-AAD+ RC as well as 7-AAD+ Treg in a representative coculture performed in the presence of OKT3 and rIL-2 (1000 IU/ml rIL-2 vs 150 IU/ml rIL-2). B, Percentages of 7-AAD+ Treg and RC following cocultures in the presence of different IL-2 concentrations of these cells obtained from PBMC of 10 HNSCC patients. C, Suppression of RC proliferation in response to OKT3 in cocultures of Treg and CD4+ RC obtained from 10 HNSCC patients. The data in B and C are means ± SD. Cell death was measured in FLOCA, and proliferation inhibition in CFSE-based assays is as described in Materials and Methods. Note that IL-2 at high concentrations (1000 IU/ml) protects Treg from RC-mediated death.
FIGURE 4
FIGURE 4
The importance of cell-to-cell contact in Treg-mediated suppression and apoptosis. A, Suppression of CD8+ RC and their death in the presence of IL-2 are cell contact-dependent. In cocultures of Treg with autologous CD8+ RC, suppression of RC proliferation was completely inhibited in the presence of Tw and 1000 IU/ml or 150 IU/ml rIL-2. Death (% 7-AAD+) of CD8+ RC was also inhibited in the presence of Tw, although only partial inhibition was seen in the presence of 1000 IU/ml IL-2. Results are means ± SD from three experiments performed with CD8+ RC/Treg cocultures. B, Suppression of CD4+ RC proliferation is cell contact-dependent in the presence of rIL-2 and so is their apoptosis; however, CD4+ RC apoptosis is only partly inhibited in the Tw system in the presence of 1000 IU/ml IL-2. Results are means ± SD from three experiments performed with CD4+ RC/Treg cocultures. The T cell subsets used in these experiments were isolated from patients with HNSCC.
FIGURE 5
FIGURE 5
Expression of Fas and FasL on human Treg and CD4+ or CD8+ RC. A, Freshly harvested or OKT3- and IL-2-activated CD4+ RC or CD8+ RC obtained from PBMC of a representative NC were evaluated for Fas or FasL expression by flow cytometry. B, Fresh and ex vivo-activated CD4+CD25high Treg from a representative patient with HNSCC or a representative NC were similarly tested for Fas and FasL expression. C, Histogram shows mean percentages ± SD of Fas+ and FasL+ CD4+CD25high Treg in peripheral blood of all NC and HNSCC patients included in this study.
FIGURE 6
FIGURE 6
AnxV binding to Treg following ex vivo treatment of PBMC with anti-Fas agonistic CH-11 Ab. A, AnxV+ binding to fresh PBMC treated with CH-11 Ab for 4 h is shown. The gate is set on CD4+CD25high T cells. Note that Treg from a representative HNSCC patient are AnxV−, that is, they are resistant to apoptosis. B, The same experiment with OKT3 and IL-2 (150 IU/ml)-activated PBMC. Note that activated Treg from NC and HNSCC patients are resistant to Fas-mediated apoptosis. Representative data from independent experiments performed with PBMC obtained from 10 patients and 5 NC are shown.
FIGURE 7
FIGURE 7
Blocking of suppression and apoptosis mediated by Treg with FasL-blocking Ab. A, Treg mediate suppression and induce apopto-sis in autologous CD8+ RC, and this apoptosis is inhibited by a FasL-blocking Ab NOK-1. B, Treg mediate suppression but do not induce apoptosis in CD4+ RC. Instead, Treg themselves undergo apoptosis in the cocultures. Death of Treg was not inhibited by NOK-1 Ab, indicating that the Fas/FasL pathway is not involved. Representative experiments of five performed with cocultures of CD8+ RC or CD4+ RC and CD4+CD25high Treg are shown. In the cocultures, cells were incubated in the presence of OKT and rIL-2 (150 IU/ml).
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