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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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Human Treg cells are induced to produce IL-17 and to downregulate FOXP3 by IL-1β. (A) “Total” CD4+CD25+ Treg cells from healthy donors were activated with anti-CD3/CD28 in medium supplemented with IL-1β, IL-2 or IL-1β+IL-2. IL-17 production was measured by ELISA and shown as mean of 12 donors. (B) A representative example of ICS for IL-17 and FOXP3 in which Treg cells were activated for 5 days with anti-CD3/CD28 together with the cytokines indicated. Data shown are from one experiment representative of five performed. (C) Cumulative data from five independent experiments showing FOXP3 staining in Treg cells activated with anti-CD3/CD28 together with cytokines (left) and negative correlation between the percentages of FOXP3+ and IL-17+ cells (right; r2 = 0.79, p < 0.05). The solid and dashed lines show the linear regression and 95% confidence interval, respectively (right). (D) A representative ICS for FOXP3 and IL-17 expression in Treg cells activated with anti-CD3/CD28 in the presence of IL-1β and IL-2, highlighting the double-positive population on day 4 (top, upper right quadrant) plus a kinetic transition through a double-positive phase (bottom). Data shown are from one experiment representative of five performed. (E) qRT-PCR for FOXP3 (left) and RORC (right) from Treg cells activated with anti-CD3/CD28 with and without cytokine. Data are normalized to unsupplemented medium; shown are mean +SD of triplicate samples and are from one experiment representative of two performed. (F) Representative Western blot showing total and phosphotylated (pY) STAT3 in Treg cells activated with anti-CD3/CD28 in the presence of the cytokines indicated. Data shown are from one experiment representative of four performed. (G) IL-17 production from Treg cells of a healthy control and two patients with hyper-IgE syndrome activated with anti-CD3/CD28 and exogenous cytokine. Data are shown as mean +SD and are from one experiment. *p < 0.05, One-way ANOVA (A), paired t-test (C, left panel), F test (C, right panel).
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fig01: Human Treg cells are induced to produce IL-17 and to downregulate FOXP3 by IL-1β. (A) “Total” CD4+CD25+ Treg cells from healthy donors were activated with anti-CD3/CD28 in medium supplemented with IL-1β, IL-2 or IL-1β+IL-2. IL-17 production was measured by ELISA and shown as mean of 12 donors. (B) A representative example of ICS for IL-17 and FOXP3 in which Treg cells were activated for 5 days with anti-CD3/CD28 together with the cytokines indicated. Data shown are from one experiment representative of five performed. (C) Cumulative data from five independent experiments showing FOXP3 staining in Treg cells activated with anti-CD3/CD28 together with cytokines (left) and negative correlation between the percentages of FOXP3+ and IL-17+ cells (right; r2 = 0.79, p < 0.05). The solid and dashed lines show the linear regression and 95% confidence interval, respectively (right). (D) A representative ICS for FOXP3 and IL-17 expression in Treg cells activated with anti-CD3/CD28 in the presence of IL-1β and IL-2, highlighting the double-positive population on day 4 (top, upper right quadrant) plus a kinetic transition through a double-positive phase (bottom). Data shown are from one experiment representative of five performed. (E) qRT-PCR for FOXP3 (left) and RORC (right) from Treg cells activated with anti-CD3/CD28 with and without cytokine. Data are normalized to unsupplemented medium; shown are mean +SD of triplicate samples and are from one experiment representative of two performed. (F) Representative Western blot showing total and phosphotylated (pY) STAT3 in Treg cells activated with anti-CD3/CD28 in the presence of the cytokines indicated. Data shown are from one experiment representative of four performed. (G) IL-17 production from Treg cells of a healthy control and two patients with hyper-IgE syndrome activated with anti-CD3/CD28 and exogenous cytokine. Data are shown as mean +SD and are from one experiment. *p < 0.05, One-way ANOVA (A), paired t-test (C, left panel), F test (C, right panel).

Mentions: It has been shown both in murine models and in man that Treg cells are plastic. In order to identify the population of Treg cells responsible for the conversion to IL-17-producing cells and the mechanisms behind this event, freshly isolated bead-enriched “whole” human Treg cells (CD4+CD25+) were initially activated in vitro in the presence of IL-1β, as previously reported [25,26], and/or IL-2. We observed that Treg cells produced IL-17 when in the presence of IL-1β and the highest levels were found when IL-2 was further added to the culture (Fig. 1A), in part due to IL-2-mediated increased expression of IL-1R1 (Supporting Information Fig. 1), as previously shown [12]. The increased IL-17 mediated by IL-1β was confirmed with intracellular staining (ICS) and was accompanied by a reduction in FOXP3 (Fig. 1B and C). A negative correlation between the percentage of IL-17 and FOXP3 single-positive cells was observed following activation of Treg cells in the presence of IL-1β plus IL-2 (r2 = 0.79, p < 0.05) (Fig. 1C). The presence of IL-2, however, maintained a higher percentage of FOXP3+ cells in ex vivo activated Treg cells but the addition of IL-1β antagonized this effect (Fig. 1C). The conversion of Treg cells to Th17 cells, rather than outgrowth of a contaminating population, was suggested by repeated observation of cells that were double positive for both FOXP3 and IL-17 (Fig. 1D, top panel), plus a kinetic transition through a double-positive phase (Fig. 1D, lower panel and Supporting Information Fig. 2), indicative of a transitional stage. In the double-positive population, FOXP3 levels were lower in those cells expressing higher IL-17 levels (Fig. 1D).


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)

Human Treg cells are induced to produce IL-17 and to downregulate FOXP3 by IL-1β. (A) “Total” CD4+CD25+ Treg cells from healthy donors were activated with anti-CD3/CD28 in medium supplemented with IL-1β, IL-2 or IL-1β+IL-2. IL-17 production was measured by ELISA and shown as mean of 12 donors. (B) A representative example of ICS for IL-17 and FOXP3 in which Treg cells were activated for 5 days with anti-CD3/CD28 together with the cytokines indicated. Data shown are from one experiment representative of five performed. (C) Cumulative data from five independent experiments showing FOXP3 staining in Treg cells activated with anti-CD3/CD28 together with cytokines (left) and negative correlation between the percentages of FOXP3+ and IL-17+ cells (right; r2 = 0.79, p < 0.05). The solid and dashed lines show the linear regression and 95% confidence interval, respectively (right). (D) A representative ICS for FOXP3 and IL-17 expression in Treg cells activated with anti-CD3/CD28 in the presence of IL-1β and IL-2, highlighting the double-positive population on day 4 (top, upper right quadrant) plus a kinetic transition through a double-positive phase (bottom). Data shown are from one experiment representative of five performed. (E) qRT-PCR for FOXP3 (left) and RORC (right) from Treg cells activated with anti-CD3/CD28 with and without cytokine. Data are normalized to unsupplemented medium; shown are mean +SD of triplicate samples and are from one experiment representative of two performed. (F) Representative Western blot showing total and phosphotylated (pY) STAT3 in Treg cells activated with anti-CD3/CD28 in the presence of the cytokines indicated. Data shown are from one experiment representative of four performed. (G) IL-17 production from Treg cells of a healthy control and two patients with hyper-IgE syndrome activated with anti-CD3/CD28 and exogenous cytokine. Data are shown as mean +SD and are from one experiment. *p < 0.05, One-way ANOVA (A), paired t-test (C, left panel), F test (C, right panel).
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fig01: Human Treg cells are induced to produce IL-17 and to downregulate FOXP3 by IL-1β. (A) “Total” CD4+CD25+ Treg cells from healthy donors were activated with anti-CD3/CD28 in medium supplemented with IL-1β, IL-2 or IL-1β+IL-2. IL-17 production was measured by ELISA and shown as mean of 12 donors. (B) A representative example of ICS for IL-17 and FOXP3 in which Treg cells were activated for 5 days with anti-CD3/CD28 together with the cytokines indicated. Data shown are from one experiment representative of five performed. (C) Cumulative data from five independent experiments showing FOXP3 staining in Treg cells activated with anti-CD3/CD28 together with cytokines (left) and negative correlation between the percentages of FOXP3+ and IL-17+ cells (right; r2 = 0.79, p < 0.05). The solid and dashed lines show the linear regression and 95% confidence interval, respectively (right). (D) A representative ICS for FOXP3 and IL-17 expression in Treg cells activated with anti-CD3/CD28 in the presence of IL-1β and IL-2, highlighting the double-positive population on day 4 (top, upper right quadrant) plus a kinetic transition through a double-positive phase (bottom). Data shown are from one experiment representative of five performed. (E) qRT-PCR for FOXP3 (left) and RORC (right) from Treg cells activated with anti-CD3/CD28 with and without cytokine. Data are normalized to unsupplemented medium; shown are mean +SD of triplicate samples and are from one experiment representative of two performed. (F) Representative Western blot showing total and phosphotylated (pY) STAT3 in Treg cells activated with anti-CD3/CD28 in the presence of the cytokines indicated. Data shown are from one experiment representative of four performed. (G) IL-17 production from Treg cells of a healthy control and two patients with hyper-IgE syndrome activated with anti-CD3/CD28 and exogenous cytokine. Data are shown as mean +SD and are from one experiment. *p < 0.05, One-way ANOVA (A), paired t-test (C, left panel), F test (C, right panel).
Mentions: It has been shown both in murine models and in man that Treg cells are plastic. In order to identify the population of Treg cells responsible for the conversion to IL-17-producing cells and the mechanisms behind this event, freshly isolated bead-enriched “whole” human Treg cells (CD4+CD25+) were initially activated in vitro in the presence of IL-1β, as previously reported [25,26], and/or IL-2. We observed that Treg cells produced IL-17 when in the presence of IL-1β and the highest levels were found when IL-2 was further added to the culture (Fig. 1A), in part due to IL-2-mediated increased expression of IL-1R1 (Supporting Information Fig. 1), as previously shown [12]. The increased IL-17 mediated by IL-1β was confirmed with intracellular staining (ICS) and was accompanied by a reduction in FOXP3 (Fig. 1B and C). A negative correlation between the percentage of IL-17 and FOXP3 single-positive cells was observed following activation of Treg cells in the presence of IL-1β plus IL-2 (r2 = 0.79, p < 0.05) (Fig. 1C). The presence of IL-2, however, maintained a higher percentage of FOXP3+ cells in ex vivo activated Treg cells but the addition of IL-1β antagonized this effect (Fig. 1C). The conversion of Treg cells to Th17 cells, rather than outgrowth of a contaminating population, was suggested by repeated observation of cells that were double positive for both FOXP3 and IL-17 (Fig. 1D, top panel), plus a kinetic transition through a double-positive phase (Fig. 1D, lower panel and Supporting Information Fig. 2), indicative of a transitional stage. In the double-positive population, FOXP3 levels were lower in those cells expressing higher IL-17 levels (Fig. 1D).

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