SUMMARY Interferon-γ (IFN-γ) promotes a population of T-bet+ CXCR3+ regulatory T (Treg) cells that limit T helper 1 (Th1) cell-mediated pathology. Our studies demonstrate that interleukin-27 (IL-27) also promoted expression of T-bet and CXCR3 in Treg cells. During infection with Toxoplasma gondii a similar population emerged which limited T cell responses and were dependent on IFN-γ in the periphery but IL-27 at mucosal sites. Transfer of Treg cells ameliorated the infection-induced pathology observed in Il27−/− mice and this was dependent on their ability to produce IL-10. Microarray analysis revealed that Treg cells exposed to either IFN-γ or IL-27 have distinct transcriptional profiles. Thus, IFN-γ and IL-27 have different roles in Treg cell biology and IL-27 is a key cytokine that promotes the development of Treg cells specialized to control Th1 cell-mediated immunity at local sites of inflammation.

SUMMARY Interleukin (IL)-27 is a key immunosuppressive cytokine that counters T helper 17 (Th17) cell-mediated pathology. To identify mechanisms by which IL-27 might exert its immunosuppressive effect, we analyzed genes in T cells rapidly induced by IL-27. We found that IL-27 priming of naïve T cells upregulated expression of programmed death ligand 1 (PD-L1) in a signal transducer and activator of transcription (STAT)1-dependent manner. When co-cultured with naïve CD4+ T cells, IL-27-primed T cells inhibited the differentiation of Th17 cells in trans through a PD-1-PD-L1 interaction. In vivo, co-administration of naïve TCR transgenic T cells (2D2 T cells) with IL-27-primed T cells expressing PD-L1 inhibited the development of Th17 cells and protected from severe autoimmune encephalomyelitis. Thus, these data identify a suppressive activity of IL-27, by which CD4+ T cells can restrict differentiation of Th17 cells in trans.

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The cytokine IL-10 has an important role in limiting inflammation in many settings, including toxoplasmosis. In these studies, an IL-10 reporter mouse was used to identify the sources of this cytokine following challenge with Toxoplasma gondii. During infection, multiple cell types expressed the IL-10 reporter but natural killer cells were a major early source of this cytokine. These IL-10 reporter+ NK cells expressed high levels of the IL-12 target genes T-bet, KLRG1, and IFN-γ, and IL-12 depletion abrogated reporter expression. However, IL-12 signaling alone was not sufficient to promote NK cell IL-10 and activation of the aryl hydrocarbon receptor (AHR) was also required for maximal IL-10 production. NK cells basally expressed the AHR, relevant chaperone proteins, and the AHR nuclear translocator (ARNT), which heterodimerizes with the AHR to form a competent transcription factor. In vitro studies revealed that IL-12 stimulation increased NK cell AHR levels, and the AHR and ARNT were required for optimal production of IL-10. Additionally, NK cells isolated from T. gondii-infected Ahr-/- mice had impaired expression of IL-10, which was associated with increased resistance to this infection. Together, these data identify the AHR as a critical cofactor involved in NK cell production of IL-10.

Inhibitory receptors (IR) are a diverse group of cell surface molecules that modulate T cell activation, but there are gaps in our knowledge of the cell-extrinsic factors that regulate their expression. The present study found that in vivo overexpression of IL-27 in mice led to increased T cell expression of PD-L1, LAG-3, TIGIT, and TIM-3. In vitro, TCR stimulation alone promoted expression of multiple IRs, whereas IL-27 alone induced expression of PD-L1. However, the combination of intermediate TCR stimulation and IL-27 resulted in synergistic induction of LAG-3, CTLA-4, and TIGIT. In vivo, infection with Toxoplasma gondii resulted in parasite-specific effector T cells that expressed high levels of IR, and at local sites of infection where IL-27 production was highest, IL-27 was required for maximal effector cell expression of PD-L1, LAG-3, CTLA-4, and TIGIT. Together, these results affirm the critical role of TCR signals in the induction of IR expression but find that during infection, IL-27 promotes T cell expression of IR. ImmunoHorizons, 2019, 3: 13-25.

Ly6C and Sca-1 (Ly6A/E) are Ly6 family GPI-anchored surface molecules that are differentially expressed by multiple immune populations. Ly6C expression has been used to distinguish short-lived effector CD4 T cells from memory precursor effector cells, whereas Sca-1 has been used in the identification of CD8 memory stem cells. This study examines the expression patterns of these molecules and establishes that, in vitro, IL-27, type I IFN, and IFN-γ are potent inducers of Ly6C and Sca-1 in naive mouse CD4 and CD8 T cells, whereas TGF-β limits their expression. The induction of Ly6C and Sca-1 by IL-27 and IFN-γ is dependent on STAT1, but not STAT3 or T-bet. In mouse splenocytes, at homeostasis, Ly6C and Sca-1 expression was not restricted to effector cells, but was also found at various levels on naive and memory populations. However, in response to infection with , pathogen-specific T cells expressed high levels of these molecules and in this context, endogenous IL-27 and IFN-γ were required for the expression of Ly6C but not Sca-1. Together, these findings highlight the TCR-dependent and cytokine-mediated signals that modulate T cell expression of Ly6C and Sca-1 in vitro and in vivo during infection.

The transcription factor T-bet has been most prominently linked to natural killer (NK) and T cell production of interferon-γ (IFN-γ), a cytokine required for the control of a diverse array of intracellular pathogens. Indeed, in mice challenged with the parasite Toxoplasma gondii, NK and T cell responses are characterized by marked increases of T-bet expression. Unexpectedly, T-bet−/− mice infected with T. gondii develop a strong NK cell IFN-γ response that controls parasite replication at the challenge site, but display high parasite burdens at secondary sites colonized by T. gondii and succumb to infection. The loss of T-bet had a modest effect on T cell production of IFN-γ but did not impact on the generation of parasite-specific T cells. However, the absence of T-bet resulted in lower T cell expression of CD11a, Ly6C, KLRG-1, and CXCR3 and fewer parasite-specific T cells at secondary sites of infection, associated with a defect in parasite control at these sites. Together, these data highlight T-bet independent pathways to IFN-γ production, and reveal a novel role for this transcription factor in coordinating the T cell responses necessary to control this infection in peripheral tissues.

GM-CSF is a potent proinflammatory cytokine that plays a pathogenic role in the CNS inflammatory disease experimental autoimmune encephalomyelitis. As IL-27 alleviates experimental autoimmune encephalomyelitis, we hypothesized that IL-27 suppresses GM-CSF expression by T cells. We found that IL-27 suppressed GM-CSF expression in CD4+ and CD8+ T cells in splenocyte and purified T cell cultures. IL-27 suppressed GM-CSF in Th1, but not Th17, cells. IL-27 also suppressed GM-CSF expression by human T cells in nonpolarized and Th1- but not Th17-polarized PBMC cultures. In vivo, IL-27p28 deficiency resulted in increased GM-CSF expression by CNS-infiltrating T cells during Toxoplasma gondii infection. Although in vitro suppression of GM-CSF by IL-27 was independent of IL-2 suppression, IL-10 upregulation, or SOCS3 signaling, we observed that IL-27–driven suppression of GM-CSF was STAT1 dependent. Our findings demonstrate that IL-27 is a robust negative regulator of GM-CSF expression in T cells, which likely inhibits T cell pathogenicity in CNS inflammation.