Regulatory T (Treg) cells are essential for maintaining peripheral tolerance, preventing autoimmune diseases and limiting chronic inflammatory diseases. However, they also limit beneficial responses by suppressing sterilizing immunity and limiting anti-tumour immunity. Given that Treg cells can have both beneficial and deleterious effects, there is considerable interest in determining their mechanisms of action. In this Review, we discuss the basic mechanisms used by Treg cells to mediate suppression, and discuss whether one or many of these mechanisms are likely to be crucial for Treg-cell function. In addition, we present the hypothesis that effector T cells may not be ‘innocent’ parties in this suppressive process and might in fact potentiate Treg-cell function.

T cell exhaustion often occurs during chronic infections and prevents optimal viral control. The molecular pathways involved in T cell exhaustion, however, remain poorly understood. We demonstrate that exhausted CD8+ T cells are subject to complex layers of negative regulation due to co-expression of multiple inhibitory receptors. Exhausted CD8+ T cells expressed up to 7 inhibitory receptors. Co-expression of multiple distinct inhibitory receptors correlated with greater T cell exhaustion and more severe infection. Regulation of T cell exhaustion by diverse inhibitory pathways was non-redundant since blockade of PD-1 and LAG-3 simultaneously in vivo synergistically improved T cell responses and reduced viral load. Thus, CD8+ T cell responses during chronic viral infections are regulated by complex patterns of co-expressed inhibitory receptors.

Regulatory T (T(reg)) 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 T(reg) 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-27beta) and interleukin-12 alpha (Il12a, which encodes IL-12alpha/p35) are highly expressed by mouse Foxp3+ (forkhead box P3) T(reg) cells but not by resting or activated effector CD4+ T (T(eff)) cells, and that an Ebi3-IL-12alpha heterodimer is constitutively secreted by T(reg) but not T(eff) cells. Both Ebi3 and Il12a messenger RNA are markedly upregulated in T(reg) cells co-cultured with T(eff) cells, thereby boosting Ebi3 and IL-12alpha production in trans. T(reg)-cell restriction of this cytokine occurs because Ebi3 is a downstream target of Foxp3, a transcription factor that is required for T(reg)-cell development and function. Ebi3-/- and Il12a-/- T(reg) 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-12alpha 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 T(reg) cells and is required for maximal suppressive activity.

Attempts to generate reliable and versatile vectors for gene therapy and biomedical research that express multiple genes have met with limited success. Here we used Picornavirus 'self-cleaving' 2A peptides, or 2A-like sequences from other viruses, to generate multicistronic retroviral vectors with efficient translation of four cistrons. Using the T-cell receptor:CD3 complex as a test system, we show that a single 2A peptide-linked retroviral vector can be used to generate all four CD3 proteins (CD3epsilon, gamma, delta, zeta), and restore T-cell development and function in CD3-deficient mice. We also show complete 2A peptide-mediated 'cleavage' and stoichiometric production of two fluorescent proteins using a fluorescence resonance energy transfer-based system in multiple cell types including blood, thymus, spleen, bone marrow and early stem cell progenitors.

Inhibitory receptors on immune cells are pivotal regulators of immune escape in cancer. Among these inhibitory receptors, CTLA-4 (targeted clinically by ipilimumab) serves as a dominant off-switch while other receptors such as PD-1 and LAG-3 seem to serve more subtle rheostat functions. However, the extent of synergy and cooperative interactions between inhibitory pathways in cancer remain largely unexplored. Here we reveal extensive co-expression of PD-1 and LAG-3 on tumor-infiltrating CD4+ and CD8+ T cells in three distinct transplantable tumors. Dual anti-LAG-3/anti-PD-1 antibody treatment cured most mice of established tumors that were largely resistant to single antibody treatment. Despite minimal immunopathological sequelae in PD-1 and LAG-3 single knockout mice, dual knockout mice abrogated self-tolerance with resultant autoimmune infiltrates in multiple organs, leading to eventual lethality. However, Lag3−/−Pdcd1−/− mice demonstrated markedly increased survival from and clearance of multiple transplantable tumors. Together, these results define a strong synergy between the PD-1 and LAG-3 inhibitory pathways in tolerance to both self and tumor antigens. Additionally, they argue strongly that dual blockade of these molecules represents a promising combinatorial strategy for cancer.

The interleukin-12 (IL-12) family is unique in comprising the only heterodimeric cytokines, which includes IL-12, IL-23, IL-27 and IL-35. This endows these cytokines with a unique set of connections and functional interactions not shared within other cytokine families. Despite sharing many structural features and molecular partners, they mediate surprisingly diverse functional effects. Here we discuss the unique and unusual structural and functional characteristics of this cytokine family. We outline how cells might interpret seemingly similar cytokine signals to give rise to the diverse functional outcomes which characterize this cytokine family. We will also discuss the therapeutic implications of this complexity.

Regulatory T cells (Tregs) limit autoimmunity but also attenuate the magnitude of antipathogen and antitumor immunity. Understanding the mechanism of Treg function and therapeutic manipulation of Tregs in vivo requires identification of Treg-selective receptors. A comparative analysis of gene expression arrays from antigen-specific CD4(+) T cells differentiating to either an effector/memory or a regulatory phenotype revealed Treg-selective expression of LAG-3, a CD4-related molecule that binds MHC class II. Antibodies to LAG-3 inhibit suppression by induced Tregs both in vitro and in vivo. Natural CD4(+)CD25(+) Tregs express LAG-3 upon activation, which is significantly enhanced in the presence of effector cells, whereas CD4(+)CD25(+) Tregs from LAG-3(-/-) mice exhibit reduced regulatory activity. Lastly, ectopic expression of LAG-3 on CD4(+) T cells significantly reduces their proliferative capacity and confers on them suppressor activity toward effector T cells. We propose that LAG-3 marks regulatory T cell populations and contributes to their suppressor activity.

Regulatory T cells (Tregs) play a critical role in the maintenance of immunological self-tolerance. Naïve human or murine T cell treatment with the inhibitory cytokine IL-35 induces a regulatory population, termed iTR35, that mediates suppression via IL-35, but not IL-10 or TGFβ, neither express nor require Foxp3, are strongly suppressive in five in vivo models, and exhibit in vivo stability. Treg-mediated suppression induces iTR35 generation in an IL-35- and IL-10-dependent manner in vitro, and in inflammatory conditions in vivo in Trichuris-infected intestines and within the tumor microenvironment, where they appear to contribute to the regulatory milieu. iTR35 may constitute a key mediator of infectious tolerance, may contribute to Treg-mediated tumor progression, and ex vivo generated iTR35 may possess therapeutic utility.