Functionally distinct subsets of human FOXP3+ Treg cells that phenotypically mirror effector Th cells

Abstract

FOXP3+ regulatory T (Treg) cells are a broadly acting and potent anti-inflammatory population of CD4+ T cells essential for maintaining immune homeostasis and preventing debilitating autoimmunity. Based on chemokine receptor expression, we identified distinct populations of Treg cells in human blood expected to colocalize with different Th cell subsets. Although each population was functionally suppressive, they displayed unique patterns of pro- and anti-inflammatory cytokine production, differentially expressed lineage-specifying transcription factors, and responded differently to antigens associated with Th1 and Th17 responses. These results highlight a previously unappreciated degree of phenotypic and functional diversity in human Treg cells that allows subsets with unique specificities and immunomodulatory functions to be targeted to defined immune environments during different types of inflammatory responses.

Figure 1

Chemokine receptor expression defines human Treg cell subsets. (A) Representative flow cytometric analysis of CCR6, CXCR3, CCR4, and CCR10 expression by gated CD4⁺CD45RO⁺CD25⁻ Th cells (top panels) and CD4⁺CD45RO⁺CD25^hiCD127^lo Treg cells (bottom panels) from peripheral blood. (B) Expression of CLA by the indicated Th and Treg cell subsets. Each symbol represents 1 donor; horizontal bars indicate the mean. Data are from 5 donors. (C) Frequency of cells expressing the indicated chemokine receptor combinations among Th and Treg cells. Each symbol represents 1 donor; horizontal bars indicate the mean. Data are from 15 donors.

Figure 2

Treg cell subsets are functionally suppressive. (A) Expression of FOXP3 by the indicated Th and Treg cell subsets. Each symbol represents 1 donor; horizontal bars indicate the mean. Data are from 9 donors. (B) Expression of Helios (left) and CTLA-4 (right) by the indicated Th and Treg cell subsets. Data are from 3 or 5 donors. (C) CFSE-labeled CD4⁺CD25⁻ responder T cells were cultured with autologous monocytes plus anti-CD3 (OKT3) in the presence or absence of the indicated sorted Treg cell subset at a 1:1 suppressor/responder ratio. Numbers indicate the frequency of proliferating CFSE^lo T cells after 5 days of culture. Data are representative of 3 independent experiments.

Figure 3

Th17- and Th1-like Treg cells coproduce pro- and anti-inflammatory cytokines. (A) Representative flow cytometric analysis of IL-10, IL-17, and IFN-γ production by sorted Th (left) and Treg (right) cell subsets stimulated for 5 hours with PMA/ionomycin. (B) Frequency of IL-17–, IL-10–, and IFN-γ–producing cells among gated FOXP3⁺ Treg cells in each of the indicated Treg cell subsets. Each symbol represents 1 donor; horizontal bars indicate the mean. Data are from 10 donors. *P < .05; ***P < .001 (ANOVA). (C) Frequency of IL-10–producing cells among IL-17⁺ cells in Th17 cells or Th17-like Treg cells (left) or among IFN-γ⁺ cells in Th1 cells or Th1-like Treg cells (right). Data are from 10 donors. ***P < .001 (2-tailed paired t test).

Figure 4

Th17- and Th1-like Treg cells express the lineage-specifying transcription factors RORγt and T-bet. (A) Quantitative RT-PCR analysis of RORγt (RORC), T-bet (TBX21), and GATA-3 (GATA3) expression by the indicated Th and Treg cell subsets. AU indicates arbitrary units. Data are means ± SEM of 7 donors. (B) Flow cytometric analysis of IFN-γ, IL-17, T-bet, and RORγt expression by sorted naive T cells (T naive), Th17-like, and Th1-like Treg cells stimulated for 5 hours with PMA/ionomycin. Data are representative of 3 independent experiments.

Figure 5

Th17- and Th1-like Treg cells are phenotypically and functionally stable. (A) Quantitative RT-PCR analysis of RORγt (RORC), T-bet (TBX21), and FOXP3 (FOXP3) expression by the indicated Th and Treg cell subsets after 9 days of in vitro expansion. AU indicates arbitrary units. Data are means ± SEM of 4 donors. (B) Flow cytometric analysis of FOXP3, IFN-γ, IL-17, and IL-10 expression on expanded Th17- and Th1-like Treg cells stimulated for 5 hours with PMA/ionomycin. Data are representative of 4 independent experiments.

Figure 6

Constitutive activation of human Treg cells. (A) Expression of ICOS (left) and Ki-67 (right) by the indicated Th and Treg cell subsets. Data are from 5 donors. (B) Flow cytometric analysis of ICOS and Ki-67 expression by the indicated Treg cell subsets. Data are representative of 4 donors. *P < .0.05; **P < .01; ***P < .001 (ANOVA).

Figure 7

C albicans– and HCMV-specific Treg cells are present in Th17- and Th1-like subsets. (A) Proportion of CFSE^lo cells (that had undergone 3 or more divisions) among the indicated Th and Treg cell subsets after 6 days of stimulation with autologous monocytes pulsed with C albicans or HCMV antigens. Each symbol represents 1 donor; small horizontal bars indicate the mean. Data are from 7 (C albicans) or 5 (HCMV) donors. (B) Flow cytometric analysis of cytokine production by gated CFSE^lo C albicans–specific Th and Treg cells after restimulation with PMA/ionomycin for 5 hours. Data are representative of 3 independent experiments using 3 different donors.