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.

SUMMARY The activation of Mixed Lineage Kinase-Like (MLKL) by Receptor Interacting Protein Kinase-3 (RIPK3) results in plasma membrane (PM) disruption and a form of regulated necrosis, called necroptosis. Here we show that during necroptosis, MLKL-dependent calcium (Ca++) influx and phosphatidylserine (PS) exposure on the outer leaflet of the plasma membrane preceded loss of PM integrity. Activation of MLKL results in the generation of broken, PM “bubbles” with exposed PS that are released from the surface of the otherwise intact cell. The ESCRT-III machinery is required for formation of these bubbles, and acts to sustain survival of the cell when MLKL activation is limited or reversed. Under conditions of necroptotic cell death, ESCRT-III controls the duration of plasma membrane integrity. As a consequence of the action of ESCRT-III, cells undergoing necroptosis can express chemokines and other regulatory molecules, and promote antigenic cross-priming of CD8+ T cells.

Regulatory T cells (Tregs) play a crucial role in the immune system by preventing autoimmunity, limiting immunopathology, and maintaining immune homeostasis1. However, they also represent a major barrier to effective anti-tumor immunity and sterilizing immunity to chronic viral infections1. The transcription factor Foxp3 plays a major role in the development and programming of Treg cells2,3. The relative stability of Tregs at inflammatory disease sites has been highly contentious4-6. There is considerable interest in identifying pathways that control Treg stability as many immune-mediated diseases are characterized by either exacerbated or limited Treg function. Here we show that the immune cell-expressed ligand semaphorin-4a (Sema4a) and the Treg-expressed receptor neuropilin-1 (Nrp1) interact to potentiate Treg function and survival in vitro and in inflammatory sites in vivo. Nrp1 is dispensable for suppression of autoimmunity and maintenance of immune homeostasis, but is required by Tregs to limit anti-tumor immune responses and to cure established inflammatory colitis. Sema4a ligation of Nrp1 restrained Akt phosphorylation cellularly and at the immunologic synapse (IS) via phosphatase and tensin homolog (PTEN), which increased nuclear localization of the transcription factor Foxo3a. The Nrp1-induced transcriptome promoted Treg stability by enhancing quiescence/survival factors while inhibiting programs that promote differentiation. Importantly, this Nrp1-dependent molecular program is evident in intratumoral Tregs. Our data support a model in which Treg stability can be subverted in certain inflammatory sites, but is maintained by a Sema4a:Nrp1 axis, highlighting this pathway as a potential therapeutic target that could limit Treg-mediated tumor-induced tolerance without inducing autoimmunity.

Regulatory T (Treg) cells respond to immune and inflammatory signals to mediate immunosuppression, but how functional integrity of Treg cells is maintained under activating environments remains elusive. Here we found that autophagy was active in Treg cells and supported their lineage stability and survival fitness. Treg cell-specific deletion of the essential autophagy gene Atg7 or Atg5 led to loss of Treg cells, increased tumor resistance, and development of inflammatory disorders. Atg7-deficient Treg cells had increased apoptosis and readily lost Foxp3 expression, especially after activation. Mechanistically, autophagy deficiency upregulated mTORC1 and c-Myc function and glycolytic metabolism that contributed to defective Treg function. Therefore, autophagy couples environmental signals and metabolic homeostasis to protect lineage and survival integrity of Treg cells in activating contexts.

Defects in dying cell clearance are postulated to underlie the pathogenesis of systemic lupus erythematosus (SLE)1. Mice lacking molecules associated with dying cell clearance develop SLE-like disease2, and phagocytes from SLE patients often display defective clearance and increased inflammatory cytokine production when exposed to dying cells in vitro. Previously, we3–6 and others7 described a form of noncanonical autophagy called “LC3-associated phagocytosis” (LAP), wherein phagosomes containing engulfed particles, including dying cells3,4,7, recruit elements of the autophagy pathway to facilitate phagosome maturation and digestion of cargo. Genome-wide association studies have identified polymorphisms in atg58 and possibly atg79, involved in both canonical autophagy and LAP3–7, as predisposition markers for SLE. Here, we describe the consequences of defective LAP in vivo. Mice lacking any of several components of the LAP pathway display elevated serum inflammatory cytokines, autoantibodies, glomerular immune complex deposition, and evidence of kidney damage. Dying cells, injected into LAP-deficient animals, are engulfed but not efficiently degraded, and trigger acute elevation of pro-inflammatory cytokines but not the anti-inflammatory interleukin (IL)-10. Repeated injection of dying cells into LAP-deficient, but not LAP-sufficient animals accelerated SLE-like disease, including increased serum levels of autoantibodies. In contrast, animals deficient for genes required for canonical autophagy but not LAP do not display defective dead cell clearance, inflammatory cytokine production, or SLE-like disease, and like wild-type animals, produce IL-10 in response to dying cells. Therefore, defects in LAP, rather than canonical autophagy, can cause SLE-like phenomena, and may contribute to the pathogenesis of SLE.