tTregs, pTregs, and iTregs: similarities and differences

Abstract

Foxp3(+) T-regulatory cells (Tregs) are primarily generated in the thymus (tTreg), but also may be generated extrathymically at peripheral sites (pTreg), or induced in cell culture (iTreg) in the presence of transforming growth factor β (TGFβ). A major unresolved issue is how these different populations of Tregs exert their suppressive function in vivo. We have developed novel systems in which the function of Tregs can be evaluated in vivo in normal mice. Our studies demonstrate that one prominent mechanism of action of polyclonal tTregs is to inhibit T-effector cell trafficking to the target organ, while antigen-specific iTregs primarily prevent T-cell priming by acting on antigen-presenting dendritic cells (DCs). Interleukin-10 (IL-10) plays an important role in the suppressive function of antigen-specific iTregs by controlling the expression of MARCH1 and CD83 on the DC. Activated tTregs may mediate infectious tolerance by delivery of cell surface-expressed TGFβ to naive responder T cells to generate pTregs. Manipulation of Treg function will require the ability to differentiate tTregs from pTregs and iTregs. The expression of the transcription factor Helios has proven to be a useful marker for the identification of stable tTregs in both mouse and human.

Keywords: Helios; IL-10; Treg cells; infectious tolerance; organ-specific autoimmunity; stability.

Fig. 1. Polyclonal tTregs and antigen-specific iTregs differentially modulate T-effector cell expansion in vivo

Naive antigen-specific TCR Tg T cells were co-transferred with polyclonal tTregs (left panel) or iTregs specific for the same antigen (right panel) into normal mice. One day later, the mice were immunized with the cognate peptide, and the number of antigen-specific T effectors in the draining lymph node quantitated 4 days later. Polyclonal tTregs augmented the number of T effectors compared to non-treated mice, while antigen-specific iTregs markedly suppressed the number of T effectors recovered.

Fig. 2. Antigen-specific iTregs disable the ability of DCs to present antigen

Antigen-pulsed DCs were cultured for 18 h alone or in the presence of activated antigen-specific T-effector cells or antigen-specific iTregs. The DCs were then separated from the T cells by cell sorting and co-cultured for 72 h with naive antigen-specific T cells. The capacity of the DCs to activate T cells was quantitated by the extent of dilution of CFSE by the T effectors.

Fig. 3. Proposed model for antigen-specific iTreg-mediated downregulation of the expression of MHC class II peptide complexes

iTregs recognize their cognate antigen via their TCR and locally deliver IL-10 resulting in ubiquitination and degradation of MHC class II- cognate peptide complexes, while leaving complexes lacking the specific peptide intact on the cell surface.

Fig. 4. A comparison of the expression of Helios and Nrp1 in Foxp3⁺ and Foxp3⁻ T cells

Lymphocytes from the indicated tissues of 8-week-old C57BL/6 mice were stained for CD4 and Nrp1 and then analyzed for expression of Foxp3 and Helios by intracellular staining. Helios and Nrp1 expression are shown when gated on CD4⁺Foxp3⁻ cells (top) or CD4⁺Foxp3⁺ cells (bottom).