Identification of T helper type 1-like, Foxp3+ regulatory T cells in human autoimmune disease

Abstract

CD4(+)CD25(high)CD127(low/-) forkhead box p3 (Foxp3)(+) regulatory T cells (T(reg) cells) possess functional plasticity. Here we describe a higher frequency of T helper type 1 (T(H)1)-like, interferon-γ (IFN-γ)-secreting Foxp3(+) T cells in untreated subjects with relapsing remitting multiple sclerosis (RRMS) as compared to healthy control individuals. In subjects treated with IFN-β, the frequency of IFN-γ(+)Foxp3(+) T cells is similar to that in healthy control subjects. In vitro, human T(reg) cells from healthy subjects acquire a T(H)1-like phenotype when cultured in the presence of interleukin-12 (IL-12). T(H)1-like T(reg) cells show reduced suppressive activity in vitro, which can partially be reversed by IFN-γ-specific antibodies or by removal of IL-12.

Figure 1

T_reg cells from individuals with RRMS secrete IFN-γ ex vivo. (a) The frequency of FACS-sorted IFN-γ⁺ and IL-17⁺ T_reg cells in healthy control individuals (left) and untreated individuals with RRMS (middle, n = 17) gated on Foxp3⁺ T_reg cells. Right, purity analysis of the sorted IFN-γ⁺Foxp3⁺ and IFN-γ Foxp3⁺ populations from subjects with RRMS used for methylation analysis in c. (b) Percentage of IFN-γ⁺Foxp3⁺ and IL-17⁺Foxp3⁺ T_reg cells (n = 17) as a proportion of total Foxp3⁺ T_reg cells. (c) Representative example of methylation analysis of the TSDR region of the FOXP3 locus in sorted IFN-γ⁺Foxp3⁺ and IFN-γ⁻ Foxp3⁺ T_reg cells from subjects with RRMS. An analysis of IFN-γ⁺Foxp3⁻ memory T cells from subjects with RRMS is shown as a control. (d) Proliferation of responder T (T_resp) cells cultured with ex vivo FACS-sorted T_reg cells from healthy control subjects and untreated subjects with multiple sclerosis (MS; T_reg cell:T_resp cell ratio of 1:2) in the presence or absence of an IFN-γ–specific antibody (n = 4). (e) The frequency of IFN-γ⁺ and IL-17⁺ T_reg cells in healthy control subjects (left) or IFN-β–treated patients with RRMS (right) as assessed by intracellular cytokine staining and FACS analysis. The bar diagram (right) shows the percentage of IFN-γ⁺Foxp3⁺ and IL-17⁺Foxp3⁺ cells as a proportion of total Foxp3⁺ T_reg cells in healthy controls or IFN-β–treated patients with RRMS (n = 12). Approval for studies was obtained from the Brigham and Women’s Hospital Institutional Review Board, and informed consent was obtained from all donors.

Figure 2

Characterization of IL-12–driven, IFN-γ⁺Foxp3⁺ T_reg cells in vitro from healthy controls. (a) Intracellular staining for IFN-γ, IL-4, IL-17 and IL-10 of untreated (upper row) and IL-12-stimulated (bottom row) human T_reg cells from healthy controls at day 4. (b) mRNA expression of FOXP3 (left), GATA3 (middle) and TBX21 (right) in T_reg cells stimulated in the presence or absence of IL-12 for 5 d (data are a representative example of three experiments performed with similar results; *P < 0.05). (c) Proliferation of T_resp cells cocultured for 3 d with T_reg cells (T_reg cell:T_resp cell ratio of 1:2) previously treated with IL-2 (top) or IL-2 + IL-12 (bottom), as assessed by carboxyfluorescein succinimidyl ester (CFSE) dilution. Histograms depict unstimulated T_resp cells alone (top left), T_resp cells stimulated with antibody to CD3 (anti-CD3) and antigen-presenting cells without T_reg cells (bottom left) and T_resp cell and T_reg cell cocultures without blocking antibodies (second column), in the presence of an IL-10–specific blocking antibody (anti–IL-10; third column), IFN-γ–specific blocking antibody (anti–IFN-γ; fourth column) or both antibodies (fifth column). (d) Representative example of staining for T_H1-associated chemokines(CCR5 and CXCR3) and TGF-β on IFN-γ⁺Foxp3⁺ and IFN-γ⁻ Foxp3⁺ T_reg cells (data are representative of three experiments).