Lauren W. Collison

The inhibitory cytokine IL-35 contributes to regulatory T-cell function

Regulatory T (Treg) cells are a critical sub-population of CD4+ T cells that are essential for maintaining self tolerance and preventing autoimmunity, for limiting chronic inflammatory diseases, such as asthma and inflammatory bowel disease, and for regulating homeostatic lymphocyte expansion. However, they also suppress natural immune responses to parasites and viruses as well as anti-tumour immunity induced by therapeutic vaccines. Although the manipulation of Treg function is an important goal of immunotherapy, the molecules that mediate their suppressive activity remain largely unknown. Here we demonstrate that Epstein-Barr-virus-induced gene 3 (Ebi3, which encodes IL-27β) and interleukin-12 alpha (Il12a, which encodes IL-12α/p35) are highly expressed by mouse Foxp3+ (forkhead box P3) Treg cells but not by resting or activated effector CD4+ T (Teff) cells, and that an Ebi3–IL-12α heterodimer is constitutively secreted by Treg but not Teff cells. Both Ebi3 and Il12a messenger RNA are markedly upregulated in Treg cells co-cultured with Teff cells, thereby boosting Ebi3 and IL-12α production in trans. Treg-cell restriction of this cytokine occurs because Ebi3 is a downstream target of Foxp3, a transcription factor that is required for Treg-cell development and function. Ebi3–/– and Il12a–/– Treg cells have significantly reduced regulatory activity in vitro and fail to control homeostatic proliferation and to cure inflammatory bowel disease in vivo. Because these phenotypic characteristics are distinct from those of other IL-12 family members, this novel Ebi3–IL-12α heterodimeric cytokine has been designated interleukin-35 (IL-35). Ectopic expression of IL-35 confers regulatory activity on naive T cells, whereas recombinant IL-35 suppresses T-cell proliferation. Taken together, these data identify IL-35 as a novel inhibitory cytokine that may be specifically produced by Treg cells and is required for maximal suppressive activity.

The composition and signaling of the IL-35 receptor are unconventional

Interleukin 35 (IL-35) belongs to the IL-12 family of heterodimeric cytokines but has a distinct functional profile. IL-35 suppresses T cell proliferation and converts naive T cells into IL-35-producing induced regulatory T cells (iTr35 cells). Here we found that IL-35 signaled through a unique heterodimer of receptor chains IL-12Rβ2 and gp130 or homodimers of each chain. Conventional T cells were sensitive to IL-35-mediated suppression in the absence of one receptor chain but not both receptor chains, whereas signaling through both chains was required for IL-35 expression and conversion into iTr35 cells. Signaling through the IL-35 receptor required the transcription factors STAT1 and STAT4, which formed a unique heterodimer that bound to distinct sites in the promoters of the genes encoding the IL-12 subunits p35 and Ebi3. This unconventional mode of signaling, distinct from that of other members of the IL-12 family, may broaden the spectrum and specificity of IL-35-mediated suppression.

REGULATORY T CELL SUPPRESSION IS POTENTIATED BY TARGET T CELLS IN A CELL CONTACT, IL-35- AND IL-10-DEPENDENT MANNER

Regulatory T cells (Treg) are believed to suppress conventional T cell (Tconv) proliferation in vitro in a contact-dependent, cytokine-independent manner, based in part on experiments in which Treg and Tconv are separated by a permeable membrane. We show that the production of IL-35, a novel inhibitory cytokine expressed by natural Treg, increases substantially following contact with Tconv. Surprisingly, Treg were able to mediate potent suppression of Tconv across a permeable membrane when placed in direct contact with Tconv in the upper chamber of a Transwell plate. Suppression was IL-35 and IL-10 dependent, and Tconv activation was required for maximal potentiation of Treg suppression. These data suggest that it is the induction of suppression, rather than the function of Treg that is obligatorily contact dependent.

Interleukin‐35: odd one out or part of the family?

Summary: Advances in cytokine biology have helped us understand the complex communication that takes place between antigen‐presenting cells and cells of the adaptive immune system, such as T cells, which collectively mediate an appropriate immune response to a plethora of pathogens while maintaining tolerance to self‐antigens. The interleukin‐12 (IL‐12) cytokine family remains one of the most important and includes IL‐12, IL‐23, IL‐27, and the recently identified IL‐35. All four are heterodimeric cytokines, composed of an α chain (p19, p28, or p35) and a β chain (p40 or Ebi3), and signal through unique pairings of five receptor chains (IL‐12Rβ1, IL‐12Rβ2, IL‐23R, gp130, and WSX‐1). Despite the interrelationship between the cytokines themselves and their receptors, their source, activity, and kinetics of expression are quite different. Studies using genetically deficient mice have greatly enhanced our understanding of the biology of these cytokines. However, interpretation of these data has been complicated by the recent realization that p40−/−, p35−/−, and Ebi3−/− mice all lack more than one cytokine (IL‐12/IL‐23, IL‐12/IL‐35, and IL‐27/IL‐35, respectively). In this review, we compare and contrast the biology of this expanded IL‐12 family and re‐evaluate data derived from the analysis of these dual cytokine‐deficient mice. We also discuss how the opposing characteristics of the IL‐12 family siblings may help to promote a balanced immune response.

The Development and Function of Regulatory T Cells

Regulatory T cells (Tregs) are a critical subset of T cells that mediate peripheral tolerance. There are two types of Tregs: natural Tregs, which develop in the thymus, and induced Tregs, which are derived from naive CD4+ T cells in the periphery. Tregs utilize a variety of mechanisms to suppress the immune response. While Tregs are critical for the peripheral maintenance of potential autoreactive T cells, they can also be detrimental by preventing effective anti-tumor responses and sterilizing immunity against pathogens. In this review, we will discuss the development of natural and induced Tregs as well as the role of Tregs in a variety of disease settings and the mechanisms they utilize for suppression.

Cutting Edge: Human Regulatory T Cells Require IL-35 To Mediate Suppression and Infectious Tolerance

Human regulatory T cells (Treg) are essential for the maintenance of immune tolerance. However, the mechanisms they use to mediate suppression remain controversial. Although IL-35 has been shown to play an important role in Treg-mediated suppression in mice, recent studies have questioned its relevance in human Treg. In this study, we show that human Treg express and require IL-35 for maximal suppressive capacity. Substantial upregulation of EBI3 and IL12A, but not IL10 and TGFB, was observed in activated human Treg compared with conventional T cells (Tconv). Contact-independent Treg-mediated suppression was IL-35 dependent and did not require IL-10 or TGF-β. Lastly, human Treg-mediated suppression led to the conversion of the suppressed Tconv into iTr35 cells, an IL-35–induced Treg population, in an IL-35–dependent manner. Thus, IL-35 contributes to human Treg-mediated suppression, and its conversion of suppressed target Tconv into IL-35–induced Treg may contribute to infectious tolerance.

In Vivo Treg Suppression Assays

Determining the activity of a regulatory T-cell population in vitro is often the first step in analyzing its function. To obtain reliable and reproducible results, it is critical to follow the protocol that is most applicable to your experimental question. We have outlined below a basic in vitro suppression assay as well as a variety of alternative/additional protocols that can be utilized alone or in combination as desired.

The plasticity of regulatory T cell function.

Regulatory T cells (Tregs) can suppress a wide variety of cell types, in diverse organ sites and inflammatory conditions. Whereas Tregs possess multiple suppressive mechanisms, the number required for maximal function is unclear. Furthermore, whether any interrelationship or cross-regulatory mechanisms exist to orchestrate and control their utilization is unknown. In this study, we assessed the functional capacity of Tregs lacking the ability to secrete both IL-10 and IL-35, which individually are required for maximal Treg activity. Surprisingly, IL-10/IL-35 double-deficient Tregs were fully functional in vitro and in vivo. Loss of IL-10 and IL-35 was compensated for by a concurrent increase in cathepsin E (Ctse) expression, enhanced TRAIL (Tnfsf10) expression, and soluble TRAIL release, rendering IL-10/IL-35 double-deficient Tregs functionally dependent on TRAIL in vitro and in vivo. Lastly, whereas C57BL/6 Tregs are normally IL-10/IL-35 dependent, BALB/c Tregs, which express high levels of cathepsin E and enhanced TRAIL expression, are partially TRAIL dependent by default. These data reveal that cross-regulatory pathways exist that control the utilization of suppressive mechanisms, thereby providing Treg functional plasticity.

Retraction: Human Regulatory T Cells Require IL-35 To Mediate Suppression and Infectious Tolerance

We wish to retract the article titled “Human Regulatory T Cells Require IL-35 To Mediate Suppression and Infectious Tolerance” by Vandana Chaturvedi, Lauren W. Collison, Clifford S. Guy, Creg J. Workman, and Dario A. A. Vignali, The Journal of Immunology , 2011, 186: [6661–6666][1]. A recent