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.

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 naïve T cells into IL-35-producing iTr35. Here we show that IL-35 signals through a unique IL-12Rβ2:gp130 heterodimer or via homodimers. Conventional T cells are sensitive to IL-35-mediated suppression in the absence of one but not both receptor chains, whereas signaling through both chains is required for IL-35 expression and iTr35 conversion. IL35R signaling requires the transcription factors STAT1 and STAT4, which form a unique heterodimer that binds to distinct sites within the Il12a and Ebi3 promoters. This unconventional mode of signaling, which is distinct from other members of the IL-12 family, may broaden the spectrum and specificity of IL-35-mediated suppression.

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Tight control of T-cell proliferation and effector function is essential to ensure an effective but appropriate immune response. Here, we reveal that this is controlled by the metalloprotease-mediated cleavage of LAG-3, a negative regulatory protein expressed by all activated T cells. We show that LAG-3 cleavage is mediated by two transmembrane metalloproteases, ADAM10 and ADAM17, with the activity of both modulated by two distinct T-cell receptor (TCR) signaling-dependent mechanisms. ADAM10 mediates constitutive LAG-3 cleavage but increases B12-fold following T-cell activation, whereas LAG-3 shedding by ADAM17 is induced by TCR signaling in a PKCh-dependent manner. LAG-3 must be cleaved from the cell surface to allow for normal T-cell activation as noncleavable LAG-3 mutants prevented proliferation and cytokine production. Lastly, ADAM10 knockdown reduced wild-type but not LAG-3 À/À T-cell proliferation. These data demonstrate that LAG-3 must be cleaved to allow efficient T-cell proliferation and cytokine production and establish a novel paradigm in which T-cell expansion and function are regulated by metalloprotease cleavage with LAG-3 as its sole molecular target.

SummaryThe physiological basis and mechanistic requirement for the high immunoreceptor tyrosine activation motifs (ITAM) multiplicity of the T cell receptor (TCR)-CD3 complex remains obscure. Here we show that while low TCR-CD3 ITAM multiplicity is sufficient to engage canonical TCR-induced signaling events that lead to cytokine secretion, high TCR-CD3 ITAM multiplicity is required for TCR-driven proliferation. This is dependent on compact immunological synapse formation, interaction of the adaptor Vav1 with phosphorylated CD3 ITAMs to mediate Notch1 recruitment and activation and ultimately c-Myc-induced proliferation. Analogous mechanistic events are also required to drive proliferation in response to weak peptide agonists. Thus, the TCR-driven pathways that initiate cytokine secretion and proliferation are separable and co-ordinated by the multiplicity of phosphorylated TCR-CD3 ITAMs.

The need for reliable, multicistronic vectors for multigene delivery is at the forefront of biomedical technology. This article describes the design and construction of 2A peptide-linked multicistronic vectors, which can be used to express multiple proteins from a single open reading frame (ORF). The small 2A peptide sequences, when cloned between genes, allow for efficient, stoichiometric production of discrete protein products within a single vector through a novel "cleavage" event within the 2A peptide sequence. Expression of more than two genes using conventional approaches has several limitations, most notably imbalanced protein expression and large size. The use of 2A peptide sequences alleviates these concerns. They are small (18-22 amino acids) and have divergent amino-terminal sequences, which minimizes the chance for homologous recombination and allows for multiple, different 2A peptide sequences to be used within a single vector. Importantly, separation of genes placed between 2A peptide sequences is nearly 100%, which allows for stoichiometric and concordant expression of the genes, regardless of the order of placement within the vector.

Highlights d Treg cells shape the intratumoral immune landscape required for tumor immune evasion d Treg cells promote an M2-like TAM phenotype indirectly by suppressing CD8 + T cells d CD8 + T cell-derived IFNg blocks SREBP1-dependent fatty acid metabolism in TAMs d SREBP1 inhibition augments the efficacy of immune checkpoint blockade