Differential control of Helios(+/-) Treg development by monocyte subsets through disparate inflammatory cytokines

Abstract

Foxp3(+) regulatory T cells (Tregs) play a pivotal role in control of autoimmunity and pathological immune responses. Helios, the Ikarus family transcription factor, binds to the Foxp3 promoter, stabilizing its expression, and is expressed in 70% of peripheral Tregs of healthy individuals. This frequency is altered during malignancy, infection, and autoimmunity, although the mechanisms that control proliferation and relative numbers of Helios(+/-) Tregs remain largely unknown. Using a T-cell-monocyte in vitro stimulation assay, we now show that proliferation of Helios(+) Tregs is inhibited by CD16(+) monocyte subset. Antibody blocking with anti-interleukin (IL)-12 reversed this inhibition, whereas addition of IL-12 suppressed Helios(+) Treg expansion, indicating that CD16(+) monocyte control of Helios(+) Treg numbers is mediated through IL-12. In contrast, proliferation of Helios(-) Tregs, which express higher levels of tumor necrosis factor receptor II (TNFRII), was suppressed by TNF-α, whereas anti-TNF-α and anti-TNFRII reversed the inhibition. CD16(-) monocyte subset was mainly responsible for TNF-α-mediated control of Helios(-) Treg expansion. Altogether, these data suggest a differential role for monocyte subsets in control of Helios(+/-) Treg development that is mediated by distinct inflammatory cytokines. These data may have important implications for understanding the pathogenesis as well as control of chronic inflammatory and autoimmune diseases.

Figure 1

Helios^+/− Treg proliferation is differentially controlled by monocyte subsets. Autologous CD3⁺ T cells, CD14⁺CD16^- and CD16⁺ monocyte subsets were purified from PBMCs of healthy volunteer controls. T cells were CFSE-labeled and co-cultured with CD14⁺CD16⁻ cells with or without CD16⁺ monocytes (as indicated by + and −) in the presence of anti-CD3 for 7 days. (A) The gating strategy to analyze the frequency of Foxp3^hi in divided (CFSE^lo) CD4⁺ subset is shown. (B) Representative dot plot of Helios and Foxp3 expression gated on divided CFSE^lo CD4⁺ cells. Gating strategy for cells expressing Foxp3^hiHelios⁺ and Foxp3^hiHelios⁻ is indicated. (C) The percentage of Foxp3^hi in CFSE^lo CD4⁺ subset (“Total Tregs”) as well as Foxp3^hiHelios⁺ (“Helios⁺ Tregs”) and Foxp3^hiHelios⁻ (“Helios⁻ Tregs”) before and after addition of CD16⁺ monocytes is shown. The P value was calculated by paired t-test and indicates that addition of CD16⁺ cells decreases Treg development in healthy normal volunteers (P = .021), as per our previously published data, as well as Helios⁺ Treg development (P = .015), but has a less obvious (not statistically significantly) effect on Helios⁻ Treg proliferation.

Figure 2

Helios⁺ Treg proliferation is inhibited by CD16⁺ monocytes through IL-12. (A) Purified T cells were co-cultured with purified autologous CD14⁺CD16⁻ cells without or with CD16⁺ monocytes in the presence of neutralizing anti–IL-12 or isotype control (“IgG”) antibodies, and after 7 days of stimulation with anti-CD3, the frequencies of Foxp3^hi (“Total Tregs”) as well as Foxp3^hiHelios⁺ (“Helios⁺ Tregs”) and Foxp3^hiHelios⁻ (“Helios⁻ Tregs”) in CFSE^loCD4⁺ subset were analyzed. The P value was calculated by paired t-test comparing the effects of treatment with anti–IL-12 relative to IgG control. In the presence of CD16⁺ cells, IL-12 neutralization increased total Treg and Helios⁺ Treg subset proliferation, but not the proliferation of Helios⁻ Treg, indicating that CD16⁺ monocytes inhibit Helios⁺ Treg development through IL-12. (B) IL-12 was added at the start of the co-cultures of T cells and monocytes, which were stimulated with anti-CD3 for 7 days. Treg (total, Helios⁺, and Helios⁻) proliferation was then compared in the absence (“Medium”) or presence (“IL-12”) of cytokine by paired t-test. The calculated P values indicate that total Treg and Helios⁺, but not Helios⁻, Treg proliferation, are inhibited by IL-12.

Figure 3

TNF-α inhibits Helios⁻ Treg proliferation through TNFRII. (A) Purified T cells were co-cultured with purified monocytes in the presence of neutralizing anti–TNF-α antibody or an isotype control (“IgG”) antibody, and after 7 days of stimulation with anti-CD3, the frequencies of Foxp3^hi (“Total Tregs”) as well as Foxp3^hiHelios⁺ (“Helios⁺ Tregs”) and Foxp3^hiHelios⁻ (“Helios⁻ Tregs”) in theCFSE^lo CD4⁺ subset were analyzed. The P value was calculated by paired t-test comparing the effects of treatment with anti–TNF-α relative to IgG control and indicates that Helios⁺ Treg proliferation is reduced, whereas Helios⁻ Treg proliferation is increased when TNF-α is neutralized through anti–TNF-α. (B) TNF-α was added at the start of the co-cultures of T cells and monocytes stimulated with anti-CD3 for 7 days. Treg (total, Helios⁺, and Helios⁻) proliferation was then compared in the absence (“Medium”) or presence (“TNF-α”) of cytokine by paired t-test. The calculated P values indicate that only Helios⁻ Treg proliferation is targeted by TNF-α, causing a decrease in proliferation of this subset. (C) Co-cultures of T cells and monocytes were treated with anti-TNFRI or (D) anti-TNFRII and stimulated with anti-CD3 for 7 days. Treg (total, Helios⁺, and Helios⁻) proliferation was then compared with IgG control antibody by paired t-test. The calculated P values indicate that only blocking with anti-TNFRII affects proliferation of Helios⁻ Tregs. (E) Percent increase in the numbers of Tregs (total, Helios⁺, and Helios⁻) in the presence of anti-TNFRI and anti-TNFRII relative to IgG control is shown for the studies presented in (C) and (D) and is consistent with a role for TNFRII, but not TNFRI, in Helios⁻ Treg development. (F) Representative dot plot of TNFRII and Helios expression in divided Foxp3^hi Tregs, showing surface expression levels of TNFRII on Helios^+/− Tregs. (G) Relative mean fluorescence intensity analysis of TNFRII expression on Helios^+/− Tregs is shown, indicating that Helios⁻ Tregs express higher levels of TNFRII compared with Helios⁺ Tregs.

Figure 4

CD16⁻ monocytes control TNF-α–mediated Helios⁻ Treg proliferation. Purified T cells were co-cultured with purified autologous CD14⁺CD16⁻ cells without or with CD16⁺ monocytes in the presence of neutralizing (A) anti–TNF-α or (B) anti-TNFRII, and after 7 days of stimulation with anti-CD3, the frequencies of Foxp3^hi (“Total Tregs”) as well as Foxp3^hiHelios⁺ (“Helios⁺ Tregs”) and Foxp3^hiHelios⁻ (“Helios⁻ Tregs”) in the CFSE^lo CD4⁺ subset were analyzed. The P value was calculated by paired t-test comparing the effects of treatment with neutralization antibody relative to IgG control. In co-cultures without CD16⁺ cells, anti–TNF-α treatment decreased Helios⁺ proliferation but increased Helios⁻ Treg expansion, similar to data obtained when total monocytes were used. In the presence of CD16⁺ monocytes, Helios⁻ Tregs were expanded relative to IgG control. (B) The same assays were performed as in (A) except that for TNF-α blockade, anti-TNFRII was used instead of anti–TNF-α. In co-cultures without CD16⁺ cells, anti-TNFRII treatment only affected Helios⁻ Treg proliferation as indicated by P values, similar to data obtained when total monocytes (Figure 3A) were used.

Figure 5

Helios^+/− Treg numbers are not affected by antibody blocking with anti– IL-6 or anti–IL-10 in T cell–monocyte co-cultures. Relative change compared with IgG control in (A) total Treg (“Total Tregs”) as well as (B) Helios⁺ (“Helios⁺ Tregs”) and (C) Helios^– (“Helios⁻ Tregs”) Treg numbers in T cell co-cultured with total monocytes or monocyte subsets (CD14⁺CD16⁻ cells without or with CD16+ monocytes) treated with neutralizing antibodies to IL-6 and IL-10 are shown. For comparison, the relative change in Treg numbers after treatment with anti–IL-12 and anti-TNF-α is also shown.

Figure 6

Hypothetical model of differential regulation of Helios^+/− Treg development by monocyte subsets. Interaction of T effector (Teff) with CD16⁻ monocyte subset results in expression of TNF-α, which in turn inhibits Helios⁻ Treg development, whereas T cell–CD16⁺ monocyte interactions trigger IL-12 expression, which inhibits Helios⁺ Treg proliferative responses.