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CD161 expression characterizes a subpopulation of human regulatory T cells that produces IL-17 in a STAT3-dependent manner.

Afzali B, Mitchell PJ, Edozie FC, Povoleri GA, Dowson SE, Demandt L, Walter G, Canavan JB, Scotta C, Menon B, Chana PS, Khamri W, Kordasti SY, Heck S, Grimbacher B, Tree T, Cope AP, Taams LS, Lechler RI, John S, Lombardi G - Eur. J. Immunol. (2013)

Bottom Line: Importantly, we find that IL-17 production is STAT3 dependent, with Treg cells from patients with STAT3 mutations unable to make IL-17.Finally, we show that CD161(+) population III Treg cells accumulate in inflamed joints of patients with inflammatory arthritis and are the predominant IL-17-producing Treg-cell population at these sites.As IL-17 production from this Treg-cell subpopulation is not accompanied by a loss of regulatory function, in the context of cell therapy, exclusion of these cells from the cell product may not be necessary.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Centre for Transplantation, King's College London, King's Health Partners, Guy's Hospital, London, UK. susan.john@kcl.ac.uk

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Population III CD161+ Treg cells are the population with “IL-17 potential”. (A) The gating strategy, adapted from Miyara et al. [32], used to identify populations I, II, and III CD4+CD25hiCD127lo Treg cells is shown. A representative example from multiple experiments is shown (please see Supporting Information Fig. 4 for full gating pathway). (B) The IL-17 concentrations in supernatants of populations I, III, and II Treg cells activated with anti-CD3/CD28 coated beads in the presence of medium only (medium) or medium supplemented with IL-1β and IL-2 (IL-1β+IL-2) for 5 days were determined by ELISA. Data are shown as mean + SD pooled from three experiments using blood from three independent healthy donors. (C, D) CD161 expression on populations I, III, and II Treg cells was determined by flow cytometry, (C) a representative example and (D) the mean + SD of five donors is shown. (E) IL-17 production by population I CD161−, population III CD161−, and population III CD161+ Treg cells activated with anti-CD3/CD28 coated beads in the presence of medium alone (medium) or medium supplemented with IL-1β and IL-2 (IL-1β+IL-2) for 5 days are shown as mean +SD of data pooled from five experiments performed * p < 0.05, paired t-test. (F) RORγt and IL-17 expression in population I CD161−, population III CD161−, and population III CD161+ Treg cells after 5 days activation with anti-CD3/CD28 coated beads and IL-1β+IL-2 were determined by flow cytometry. Values in the gates are percentages. Data shown are representative of two independent experiments performed. (G) qRT-PCR showing fold induction of RORC (top) and IRF-4 (bottom) in population I CD161−, population III CD161−, and population III CD161+ Treg cells activated with anti-CD3/CD28 coated beads in the presence of medium alone (medium) or medium supplemented with IL-1β and IL-2 (IL-1β+IL-2) for 5 days. The panels show individual data from three independent healthy donors.
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fig02: Population III CD161+ Treg cells are the population with “IL-17 potential”. (A) The gating strategy, adapted from Miyara et al. [32], used to identify populations I, II, and III CD4+CD25hiCD127lo Treg cells is shown. A representative example from multiple experiments is shown (please see Supporting Information Fig. 4 for full gating pathway). (B) The IL-17 concentrations in supernatants of populations I, III, and II Treg cells activated with anti-CD3/CD28 coated beads in the presence of medium only (medium) or medium supplemented with IL-1β and IL-2 (IL-1β+IL-2) for 5 days were determined by ELISA. Data are shown as mean + SD pooled from three experiments using blood from three independent healthy donors. (C, D) CD161 expression on populations I, III, and II Treg cells was determined by flow cytometry, (C) a representative example and (D) the mean + SD of five donors is shown. (E) IL-17 production by population I CD161−, population III CD161−, and population III CD161+ Treg cells activated with anti-CD3/CD28 coated beads in the presence of medium alone (medium) or medium supplemented with IL-1β and IL-2 (IL-1β+IL-2) for 5 days are shown as mean +SD of data pooled from five experiments performed * p < 0.05, paired t-test. (F) RORγt and IL-17 expression in population I CD161−, population III CD161−, and population III CD161+ Treg cells after 5 days activation with anti-CD3/CD28 coated beads and IL-1β+IL-2 were determined by flow cytometry. Values in the gates are percentages. Data shown are representative of two independent experiments performed. (G) qRT-PCR showing fold induction of RORC (top) and IRF-4 (bottom) in population I CD161−, population III CD161−, and population III CD161+ Treg cells activated with anti-CD3/CD28 coated beads in the presence of medium alone (medium) or medium supplemented with IL-1β and IL-2 (IL-1β+IL-2) for 5 days. The panels show individual data from three independent healthy donors.

Mentions: Given the relatively small changes in transcription factor profiles observed (Fig. 1E) and the relatively low frequency of conversion seen with ICS (Fig. 1B) in “whole” bead-enriched CD4+CD25+ Treg cells, we hypothesized that IL-17 potential is restricted to one or more subpopulations of human Treg cells. Treg cells were FACS sorted into three subpopulations based on the expression of FOXP3 and CD45RA, namely CD4+CD25++CD127loCD45RA+ (population I), CD4+CD25+++CD127loCD45RA− (population II), and CD4+CD25++CD127loCD45RA− (population III), as recently published [32] (Fig. 2A and Supporting Information Fig. 4). Following stimulation in vitro in the presence of IL-1β and IL-1β+IL-2, only population III was found to produce IL-17 (Fig. 2B).


CD161 expression characterizes a subpopulation of human regulatory T cells that produces IL-17 in a STAT3-dependent manner.

Afzali B, Mitchell PJ, Edozie FC, Povoleri GA, Dowson SE, Demandt L, Walter G, Canavan JB, Scotta C, Menon B, Chana PS, Khamri W, Kordasti SY, Heck S, Grimbacher B, Tree T, Cope AP, Taams LS, Lechler RI, John S, Lombardi G - Eur. J. Immunol. (2013)

Population III CD161+ Treg cells are the population with “IL-17 potential”. (A) The gating strategy, adapted from Miyara et al. [32], used to identify populations I, II, and III CD4+CD25hiCD127lo Treg cells is shown. A representative example from multiple experiments is shown (please see Supporting Information Fig. 4 for full gating pathway). (B) The IL-17 concentrations in supernatants of populations I, III, and II Treg cells activated with anti-CD3/CD28 coated beads in the presence of medium only (medium) or medium supplemented with IL-1β and IL-2 (IL-1β+IL-2) for 5 days were determined by ELISA. Data are shown as mean + SD pooled from three experiments using blood from three independent healthy donors. (C, D) CD161 expression on populations I, III, and II Treg cells was determined by flow cytometry, (C) a representative example and (D) the mean + SD of five donors is shown. (E) IL-17 production by population I CD161−, population III CD161−, and population III CD161+ Treg cells activated with anti-CD3/CD28 coated beads in the presence of medium alone (medium) or medium supplemented with IL-1β and IL-2 (IL-1β+IL-2) for 5 days are shown as mean +SD of data pooled from five experiments performed * p < 0.05, paired t-test. (F) RORγt and IL-17 expression in population I CD161−, population III CD161−, and population III CD161+ Treg cells after 5 days activation with anti-CD3/CD28 coated beads and IL-1β+IL-2 were determined by flow cytometry. Values in the gates are percentages. Data shown are representative of two independent experiments performed. (G) qRT-PCR showing fold induction of RORC (top) and IRF-4 (bottom) in population I CD161−, population III CD161−, and population III CD161+ Treg cells activated with anti-CD3/CD28 coated beads in the presence of medium alone (medium) or medium supplemented with IL-1β and IL-2 (IL-1β+IL-2) for 5 days. The panels show individual data from three independent healthy donors.
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fig02: Population III CD161+ Treg cells are the population with “IL-17 potential”. (A) The gating strategy, adapted from Miyara et al. [32], used to identify populations I, II, and III CD4+CD25hiCD127lo Treg cells is shown. A representative example from multiple experiments is shown (please see Supporting Information Fig. 4 for full gating pathway). (B) The IL-17 concentrations in supernatants of populations I, III, and II Treg cells activated with anti-CD3/CD28 coated beads in the presence of medium only (medium) or medium supplemented with IL-1β and IL-2 (IL-1β+IL-2) for 5 days were determined by ELISA. Data are shown as mean + SD pooled from three experiments using blood from three independent healthy donors. (C, D) CD161 expression on populations I, III, and II Treg cells was determined by flow cytometry, (C) a representative example and (D) the mean + SD of five donors is shown. (E) IL-17 production by population I CD161−, population III CD161−, and population III CD161+ Treg cells activated with anti-CD3/CD28 coated beads in the presence of medium alone (medium) or medium supplemented with IL-1β and IL-2 (IL-1β+IL-2) for 5 days are shown as mean +SD of data pooled from five experiments performed * p < 0.05, paired t-test. (F) RORγt and IL-17 expression in population I CD161−, population III CD161−, and population III CD161+ Treg cells after 5 days activation with anti-CD3/CD28 coated beads and IL-1β+IL-2 were determined by flow cytometry. Values in the gates are percentages. Data shown are representative of two independent experiments performed. (G) qRT-PCR showing fold induction of RORC (top) and IRF-4 (bottom) in population I CD161−, population III CD161−, and population III CD161+ Treg cells activated with anti-CD3/CD28 coated beads in the presence of medium alone (medium) or medium supplemented with IL-1β and IL-2 (IL-1β+IL-2) for 5 days. The panels show individual data from three independent healthy donors.
Mentions: Given the relatively small changes in transcription factor profiles observed (Fig. 1E) and the relatively low frequency of conversion seen with ICS (Fig. 1B) in “whole” bead-enriched CD4+CD25+ Treg cells, we hypothesized that IL-17 potential is restricted to one or more subpopulations of human Treg cells. Treg cells were FACS sorted into three subpopulations based on the expression of FOXP3 and CD45RA, namely CD4+CD25++CD127loCD45RA+ (population I), CD4+CD25+++CD127loCD45RA− (population II), and CD4+CD25++CD127loCD45RA− (population III), as recently published [32] (Fig. 2A and Supporting Information Fig. 4). Following stimulation in vitro in the presence of IL-1β and IL-1β+IL-2, only population III was found to produce IL-17 (Fig. 2B).

Bottom Line: Importantly, we find that IL-17 production is STAT3 dependent, with Treg cells from patients with STAT3 mutations unable to make IL-17.Finally, we show that CD161(+) population III Treg cells accumulate in inflamed joints of patients with inflammatory arthritis and are the predominant IL-17-producing Treg-cell population at these sites.As IL-17 production from this Treg-cell subpopulation is not accompanied by a loss of regulatory function, in the context of cell therapy, exclusion of these cells from the cell product may not be necessary.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Centre for Transplantation, King's College London, King's Health Partners, Guy's Hospital, London, UK. susan.john@kcl.ac.uk

Show MeSH
Related in: MedlinePlus