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. 2019 Jul 15;203(2):370-378.
doi: 10.4049/jimmunol.1900388. Epub 2019 Jun 5.

Helios Deficiency Predisposes the Differentiation of CD4+Foxp3- T Cells into Peripherally Derived Regulatory T Cells

Mathias Skadow  1 , Vinay R Penna  1 , Jessica Galant-Swafford  1 , Ethan M Shevach  2 , Angela M Thornton  1
Affiliations

Helios Deficiency Predisposes the Differentiation of CD4+Foxp3- T Cells into Peripherally Derived Regulatory T Cells

Mathias Skadow et al. J Immunol. .

Abstract

The transcription factor Helios is expressed in a large percentage of Foxp3+ regulatory T (Treg) cells and is required for the maintenance of their suppressive phenotype, as mice with a selective deficiency of Helios in Treg cells spontaneously develop autoimmunity. However, mice with a deficiency of Helios in all T cells do not exhibit autoimmunity, despite the defect in the suppressor function of their Treg cell population, suggesting that Helios also functions in non-Treg cells. Although Helios is expressed in a small subset of CD4+Foxp3- and CD8+ T cells and its expression is upregulated upon T cell activation, its function in non-Treg cells remains unknown. To examine the function of Helios in CD4+Foxp3- T cells, we transferred Helios-sufficient or -deficient naive CD4+Foxp3- TCR transgenic T cells to normal recipients and examined their capacity to respond to their cognate Ag. Surprisingly, Helios-deficient CD4+ T cells expanded and differentiated into Th1 or Th2 cytokine-producing effectors in a manner similar to wild-type TCR transgenic CD4+ T cells. However, the primed Helios-deficient cells failed to expand upon secondary challenge with Ag. The tolerant state of the Helios-deficient memory T cells was not cell-intrinsic but was due to a small population of Helios-deficient naive T cells that had differentiated into Ag-specific peripheral Treg cells that suppressed the recall response in an Ag-specific manner. These findings demonstrate that Helios plays a role in the determination of CD4+ T cell fate.

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Figures

FIGURE 1.
FIGURE 1.
Helios is associated with TM and TFH cells. (A) Splenocytes from 10 wk old mice were gated on CD4+ T cells and analyzed for Foxp3 and Helios expression. Foxp3−Helios− and Foxp3− Helios+ cells were analyzed for their expression of CD44 (left) and TFH markers CXCR5 and PD-1 (right). (B) Naive CD4+ T cells (GFP−CD44loCD45RBhi) were isolated from OT-II Foxp3GFP mice and adoptively transferred into congenic F1 recipients which were immunized the following day with the indicated adjuvants. Freshly sorted cells (left) and CD45.2+ cells gated from congenic hosts on days 3 and 6 post immunization (middle and right) were analyzed for expression of Foxp3 and Helios.
FIGURE 2.
FIGURE 2.
Helios deficient mice do not display major defects. (A) Splenocytes from 4-6 mo old Ikzf2fl/fl and Ikzf2fl/fl × CD4Cre mice were gated on CD4+ T cells and analyzed for the frequency of Treg, TM, and TFH cells, as well as the number of GC B cells (B220+IgDloFas+GL7+). (B) Ikzf2fl/fl and Ikzf2fl/fl × CD4Cre mice were immunized i.p. with SRBC. Spleens were harvested 8d post immunization and analyzed for the expression of TFH markers among CD4+ Foxp3− T cells (C) and the number of GC B cells.
FIGURE 3.
FIGURE 3.
T cell activation is unaffected by Helios deficiency. (A) Purified naïve CD4+ T cells (GFP−CD44loCD45RBhi) from Ikzf2fl/fl × CD4Cre OT-II Foxp3GFP or control mice were adoptively transferred into congenic F1 recipients. Recipient mice were immunized s.c. the following day with OVA/CFA. Draining LNs were harvested on days 4, 8, and 13 post immunization and the total number of CD45.2+ OT-II cells and (B) the frequency of Teff, CD69+ and TFH cells among the OT-II cells was analyzed by FACS. (C) Naive OT-II cells were adoptively transferred into recipient mice immunized with OVA in CFA (s.c.) or OVA in alum (i.p.). The production of cytokines was assessed by ICS following a 4 h stimulation with PMA/Ionomycin. (D) Naïve OT-II cells were transferred into congenic SAP−/− mice which were immunized with OVA in alum i.p. the following day. Splenocytes were analyzed on d 8 post immunization for TFH cell differentiation of transferred OT-II cells and (E) GC B cell differentiation. (F) Serum was collected from SAP−/− mice on day 8, and OVA-specific IgG1 was measured by ELISA.
FIGURE 4.
FIGURE 4.
Helios deficient T cells cannot be reactivated. (A) Naïve CD4+ T cells were purified from Ikzf2fl/fl × CD4Cre OT-II Foxp3GFP or control mice and adoptively transferred into congenic F1 recipients. Recipient mice were immunized s.c. the following day with OVA/CFA and boosted s.c. with OVA/IFA on d10 post immunization. Draining LNs were harvested on days 10 (before boost) and days 14 and 17, and the total number of CD45.2+ OT-II cells was analyzed by FACS. (B) Experimental outline of OT-II in vitro restimulation. Naïve OT-II cells were adoptively transferred into F1 congenic mice which were immunized as before. On d4, draining LNs were pooled and CD45.2+ OT-II cells were isolated by FACS and cultured with DCs with or without OVAp (323-339). (C) After 24 h OT-II cell culture was harvested and analyzed for cell activation and cell death by FACS.
FIGURE 5.
FIGURE 5.
Helios deficient OT-II cells differentiate into pTregs. (A) Naïve CD4+ T cells from Ikzf2fl/fl × CD4Cre OT-II Foxp3GFP or control mice were adoptively transferred into congenic F1 recipients. Recipient mice were immunized s.c. the following day with OVA/CFA. Draining LNs were harvested on d11 or (B) the indicated days after immunization, gated on CD45.2+ OT-II cells and analyzed for Foxp3 expression. (C) Naïve OT-II T cells were adoptively transferred into congenic F1 recipients. Recipient mice were given OVA in drinking water ad lib. for 7 d. Foxp3 expression in CD45.2+ OT-II cells was measured in mLN and PP. (D) Naïve OT-II cells were adoptively transferred as in (A) and recipient mice were immunized s.c. the following day with OVA/IFA. Foxp3 expression in CD45.2+ OT-II cells was analyzed from the dLN on day 7.
FIGURE 6.
FIGURE 6.
Helios deficient CD4 T cells are not differentially sensitive to TGFβ, IL-2 or TCR stimulus. Purified naïve CD4+ T cells (CD25−CD44loCD45RBhi) were cultured with DCs and either (A) IL-2 plus the indicated concentrations of TGFβ or (B) neutralizing anti-mouse IL-2 (S4B6), TGFβ and the indicated concentrations of rhIL-2 or (C) neutralizing anti-mouse IL-2 (S4B6), rhIL-2, TGFβ and the indicated concentrations of soluble α-CD3. Foxp3 expression was analyzed among viable cells on d3.
FIGURE 7.
FIGURE 7.
The generation of pTreg in Helios deficient cells suppresses restimulation in an antigen specific manner. (A) Experimental outline of OT-II sequential transfer. Naïve CD45.2+ OT-II cells were adoptively transferred into congenic F1 mice (CD45.1 × CD45.2), which were immunized s.c. the next day with OVA/CFA. On d9, CD45.1+ OT-II cells were transferred into recipient mice followed by OVA/IFA immunization on day 10. (B) Draining LN were harvested on day 14 and the proliferation of CD45.2+ and CD45.1+ OT-II cells was analyzed by FACS. (C) Experimental outline of OT-II/ SMARTA sequential transfer. As in (C) CD45.1+ SMARTA cells were transferred on d9 followed by s.c. immunization with OVA + GP66-77 in IFA. (D) Draining LNs were harvested on day 14 and the proliferation of CD45.2+ OT-II cells and CD45.1+ SMARTA cells was analyzed.
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