Marko Janke

Notch regulates IL-10 production by T helper 1 cells

T helper 1 (Th1) cells mediate powerful cellular immune responses. However, if unbalanced, Th1 immunity eventually may cause pathology. Recently, it has been shown that IL-10, an antiinflammatory cytokine strongly antagonizing Th1-mediated effects, can be produced by Th1 cells and is indeed essential for self-regulation of Th1 immunity. Here, we show that Notch induces IL-10 production in newly developing and already established Th1 cells via a signal transducer and activator of transcription 4 (STAT4)-dependent process. Notch signaling in the presence of the cytokines IL-12 or IL-27 induces Th1 cells to produce large amounts of IL-10 without diminishing IFN-γ production. Notch-modified Th1 cells completely lose their inflammatory capacity and instead are able to actively suppress a Th1 cell-induced delayed-type hypersensitivity (DTH) reaction in an IL-10-dependent fashion. IL-10 production can be elicited by active forms of all four mammalian Notch receptors but was found to be specific for the Delta-like family of Notch ligands. Dendritic cells (DC) selectively acquire Delta-like 4 expression upon stimulation with various Toll-like receptor (TLR) ligands and concomitantly induce IL-10 production by Th1 cells in vitro and in vivo. This effect can be selectively reversed by pharmacological inhibitors of Notch signaling (γ-secretase inhibitor). Our data suggest that Notch regulates IL-10 production in Th1 cells by a STAT4-dependent process that converts proinflammatory Th1 cells into T cells with regulatory activity. This pathway may provide unique opportunities for therapeutic intervention in Th1-driven immune diseases and for Th1-associated vaccination strategies.

IL-2 induces in vivo suppression by CD4+CD25+Foxp3+ regulatory T cells

Interleukin‐2 (IL‐2) treatment is currently used to enhance T cell‐mediated immune responses against tumors or in viral infections. At the same time, IL‐2 is essential for the peripheral homeostasis of CD4+CD25+Foxp3+ regulatory T cells (Treg). In our study, we show that IL‐2 is also an important activator of Treg suppressive activity in vivo. IL‐2 treatment induces Treg expansion as well as IL‐10 production and increases their suppressive potential in vitro. Importantly, in vivo application of IL‐2 via gene‐gun vaccination using IL‐2 encoding DNA plasmids (pIL‐2) inhibited naive antigen‐specific T cell proliferation as well as a Th1‐induced delayed type hypersensitivity response. The suppressive effect can be transferred onto naive animals by Treg from IL‐2‐treated mice and the suppression depends on the synergistic action of IL‐10 and TGF‐β. These data highlight that during therapeutic treatment with IL‐2 the concomitant activation of Treg may indeed counteract the intended activation of cellular immunity.

Autoregulation of Th1-mediated inflammation by twist1

The basic helix-loop-helix transcriptional repressor twist1, as an antagonist of nuclear factor κB (NF-κB)–dependent cytokine expression, is involved in the regulation of inflammation-induced immunopathology. We show that twist1 is expressed by activated T helper (Th) 1 effector memory (EM) cells. Induction of twist1 in Th cells depended on NF-κB, nuclear factor of activated T cells (NFAT), and interleukin (IL)-12 signaling via signal transducer and activator of transcription (STAT) 4. Expression of twist1 was transient after T cell receptor engagement, and increased upon repeated stimulation of Th1 cells. Imprinting for enhanced twist1 expression was characteristic of repeatedly restimulated EM Th cells, and thus of the pathogenic memory Th cells characteristic of chronic inflammation. Th lymphocytes from the inflamed joint or gut tissue of patients with rheumatic diseases, Crohns disease or ulcerative colitis expressed high levels of twist1. Expression of twist1 in Th1 lymphocytes limited the expression of the cytokines interferon-γ, IL-2, and tumor necrosis factor-α, and ameliorated Th1-mediated immunopathology in delayed-type hypersensitivity and antigen-induced arthritis.

siRNA stabilization prolongs gene knockdown in primary T lymphocytes

RNA interference (RNAi)‐mediated knockdown of target gene expression represents a powerful approach for functional genomics and therapeutic applications. However, for T lymphocytes, central regulators of immunity and immunopathologies, the application of RNAi has been limited due to the lack of efficient small interfering RNA (siRNA) delivery protocols, and an inherent inefficiency of the RNAi machinery itself. Here, we use nucleofection, an optimized electroporation approach, to deliver siRNA into primary T lymphocytes with high efficiency and negligible impairment of cell function. We identify siRNA stability within the cells as the critical parameter for efficient RNAi in primary T cells. While generally short‐lived and immediately lost upon T‐cell activation when conventional siRNA is used, target gene knockdown becomes insensitive to cell activation and can persist for up to 2 wk in non‐dividing cells with siRNA stabilized by chemical modifications. Targeting CD4 and the transcription factor GATA‐3, we show that the use of stabilized siRNA is imperative for functional gene analysis during T lymphocyte activation and differentiation in vitro as well as in vivo.

Role of Blimp-1 in programing Th effector cells into IL-10 producers

The transcriptional regulator Blimp-1 is absolutely required for IL-10 production in Th1 cells and limits inflammatory effector T cell responses downstream of IL-12 and IL-27.

Persistence of effector memory Th1 cells is regulated by Hopx

Th1 cells are prominent in inflamed tissue, survive conventional immunosuppression, and are believed to play a pivotal role in driving chronic inflammation. Here, we identify homeobox only protein (Hopx) as a critical and selective regulator of the survival of Th1 effector/memory cells, both in vitro and in vivo. Expression of Hopx is induced by T‐bet and increases upon repeated antigenic restimulation of Th1 cells. Accordingly, the expression of Hopx is low in peripheral, naïve Th cells, but highly up‐regulated in terminally differentiated effector/memory Th1 cells of healthy human donors. In murine Th1 cells, Hopx regulates the expression of genes involved in regulation of apoptosis and survival and makes them refractory to Fas‐induced apoptosis. In vivo, adoptively transferred Hopx‐deficient murine Th1 cells do not persist. Consequently, they cannot induce chronic inflammation in murine models of transfer‐induced colitis and arthritis, demonstrating a key role of Hopx for Th1‐mediated immunopathology.

In vitro-induced Th17 cells fail to induce inflammation in vivo and show an impaired migration into inflamed sites

Recently, IL‐17 produced by Th17 cells was described as pro‐inflammatory cytokine with an eminent role in autoimmune diseases, e.g. rheumatoid arthritis. A lack of IL‐17 leads to amelioration of collagen‐induced arthritis. IL‐17 induction in naïve CD4+ T cells depends on IL‐6 and TGF‐β and is enhanced by IL‐23. The in vivo inflammatory potential of in vitro‐primed Th17 cells however, remains unclear. Here, we show that, although IL‐17 neutralisation results in amelioration of murine OVA‐induced arthritis, in vitro‐primed Th17 cells cannot exacerbate arthritic symptoms after adoptive transfer. Furthermore, Th17 cells cannot induce an inflammatory delayed type hypersensitivity reaction because they fail to migrate into inflamed sites, possibly due to the lack of CXCR3 expression. Also, re‐isolated Th17 cells acquired IFN‐γ expression, indicating instability of the Th17 phenotype. Taken together, the data show that IL‐6, TGF‐β and IL‐23 might not provide sufficient signals to induce “fully qualified” Th17 cells.

Liver sinusoidal endothelial cells induce immunosuppressive IL-10-producing Th1 cells via the Notch pathway.

Under homeostasis, liver sinusoidal endothelial cells (LSECs) shift intrahepatic T‐cell responses towards tolerance. However, the role of LSECs in the regulation of T‐cell‐induced liver inflammation is less clear. Here, we studied the capacity of LSECs to modulate pro‐inflammatory Th1‐cell differentiation in mice. Using in vitro co‐culture systems and subsequent cytokine analysis, we showed that LSECs induced high amounts of the anti‐inflammatory cytokine IL‐10 in developing Th1 cells. These LSEC‐stimulated Th1 cells had no pro‐inflammatory capacity in vivo but instead actively suppressed an inflammatory Th1‐cell‐induced delayed‐type hypersensitivity reaction. Blockage of IL‐10 signaling in vivo inhibited immunosuppressive activity of LSEC‐stimulated Th1 cells. We identified the Notch pathway as a mechanism how LSECs trigger IL‐10 expression in Th1 cells. LSECs expressed high levels of the Delta‐like and Jagged family of Notch ligands and induced expression of the Notch target genes hes‐1 and deltex‐1 in Th1 cells. Blockade of Notch signaling selectively inhibited IL‐10 induction in Th1 cells by LSECs. Our findings suggest that LSEC‐induced IL‐10 expression in Th1 cells via the Notch pathway may contribute to the control of hepatic inflammatory immune responses by induction of a self‐regulatory mechanism in pro‐inflammatory Th1 cells.