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Methionine-1 (M1)-linked ubiquitin chains regulate the activity of NF-κB, immune homeostasis, and responses to infection. The importance of negative regulators of M1-linked chains in vivo remains poorly understood. Here, we show that the M1-specific deubiquitinase OTULIN is essential for preventing TNF-associated systemic inflammation in humans and mice. A homozygous hypomorphic mutation in human OTULIN causes a potentially fatal autoinflammatory condition termed OTULIN-related autoinflammatory syndrome (ORAS). Four independent OTULIN mouse models reveal that OTULIN deficiency in immune cells results in cell-type-specific effects, ranging from over-production of inflammatory cytokines and autoimmunity due to accumulation of M1-linked polyubiquitin and spontaneous NF-κB activation in myeloid cells to downregulation of M1-polyubiquitin signaling by degradation of LUBAC in B and T cells. Remarkably, treatment with anti-TNF neutralizing antibodies ameliorates inflammation in ORAS patients and rescues mouse phenotypes. Hence, OTULIN is critical for restraining life-threatening spontaneous inflammation and maintaining immune homeostasis.

BACKGROUND: Differentiation of lymphocytes is frequently accompanied by cell cycle changes, interplay that is of central importance for immunity but is still incompletely understood. Here, we interrogate and quantitatively model how proliferation is linked to differentiation in CD4+ T cells. RESULTS: We perform ex vivo single-cell RNA-sequencing of CD4+ T cells during a mouse model of infection that elicits a type 2 immune response and infer that the differentiated, cytokine-producing cells cycle faster than early activated precursor cells. To dissect this phenomenon quantitatively, we determine expression profiles across consecutive generations of differentiated and undifferentiated cells during Th2 polarization in vitro. We predict three discrete cell states, which we verify by single-cell quantitative PCR. Based on these three states, we extract rates of death, division and differentiation with a branching state Markov model to describe the cell population dynamics. From this multi-scale modelling, we infer a significant acceleration in proliferation from the intermediate activated cell state to the mature cytokine-secreting effector state. We confirm this acceleration both by live imaging of single Th2 cells and in an ex vivo Th1 malaria model by single-cell RNA-sequencing. CONCLUSION: The link between cytokine secretion and proliferation rate holds both in Th1 and Th2 cells in vivo and in vitro, indicating that this is likely a general phenomenon in adaptive immunity.

Group-2 innate lymphoid cells (ILC2) play critical roles in the initiation and maintenance of type-2 immune responses, predominantly through their production of the type-2 cytokines IL-5, IL-9, and IL-13. ILC2 are essential for the efficient elimination of helminth parasites, but also contribute to the detrimental type-2 immune responses that underlie diseases such as asthma and allergy. While several transcription factors have been identified that regulate the development and function of ILC2, less is known about the post-transcriptional mechanisms that regulate these processes. We identified micro-RNAs (miRNAs) that are co-ordinately regulated in ILC2 from mice exposed to two different stimuli, namely IL-33 "alarmin" administration or Nippostrongylus brasiliensis parasitic worm infection. miR-155 is upregulated in ILC2 in response to both stimuli and miR-155-/- mice had impaired IL-33-driven ILC2 responses. Using mixed bone marrow chimeras, we demonstrate that this deficit is intrinsic to ILC2 and that miR-155 protects ILC2 from apoptosis, while having little impact on ILC2 proliferation or cytokine production. These data reveal a subset of miRNAs that are regulated upon ILC2 activation and establish a specific role for miR-155 in regulating ILC2 survival following activation.

Naive CD4+ T lymphocytes initially undergo antigen-specific activation to promote a broad-spectrum response before adopting bespoke cytokine expression profiles shaped by intercellular microenvironmental cues, resulting in pathogen-focused modular cytokine responses. Interleukin (IL)-4-induced Gata3 upregulation is important for the helper type 2 T cell (TH2 cell) polarization associated with anti-helminth immunity and misdirected allergic inflammation. Whether additional microenvironmental factors participate is unclear. Using whole mouse-genome CRISPR-Cas9 screens, we discovered a previously unappreciated role for αvβ3 integrin in TH2 cell differentiation. Low-level αvβ3 expression by naive CD4+ T cells contributed to pan-T cell activation by promoting T-T cell clustering and IL-2/CD25/STAT5 signaling. Subsequently, IL-4/Gata3-induced selective upregulation of αvβ3 licensed intercellular αvβ3-Thy1 interactions among TH2 cells, enhanced mammalian target of rapamycin (mTOR) signaling, supported differentiation and promoted IL-5/IL-13 production. In mice, αvβ3 was required for efficient, allergen-driven, antigen-specific lung TH2 cell responses. Thus, αvβ3-expressing TH2 cells form multicellular factories to propagate and amplify TH2 cell responses.