Intermittent PI3Kδ inhibition sustains anti-tumour immunity and curbs irAEs.

Phosphoinositide 3-kinase δ (PI3Kδ) has a key role in lymphocytes, and inhibitors that target this PI3K have been approved for treatment of B cell malignancies1-3. Although studies in mouse models of solid tumours have demonstrated that PI3Kδ inhibitors (PI3Kδi) can induce anti-tumour immunity4,5, its effect on solid tumours in humans remains unclear. Here we assessed the effects of the PI3Kδi AMG319 in human patients with head and neck cancer in a neoadjuvant, double-blind, placebo-controlled randomized phase II trial (EudraCT no. 2014-004388-20). PI3Kδ inhibition decreased the number of tumour-infiltrating regulatory T (Treg) cells and enhanced the cytotoxic potential of tumour-infiltrating T cells. At the tested doses of AMG319, immune-related adverse events (irAEs) required treatment to be discontinued in 12 out of 21 of patients treated with AMG319, suggestive of systemic effects on Treg cells. Accordingly, in mouse models, PI3Kδi decreased the number of Treg cells systemically and caused colitis. Single-cell RNA-sequencing analysis revealed a PI3Kδi-driven loss of tissue-resident colonic ST2 Treg cells, accompanied by expansion of pathogenic T helper 17 (TH17) and type 17 CD8+ T (TC17) cells, which probably contributed to toxicity; this points towards a specific mode of action for the emergence of irAEs. A modified treatment regimen with intermittent dosing of PI3Kδi in mouse models led to a significant decrease in tumour growth without inducing pathogenic T cells in colonic tissue, indicating that alternative dosing regimens might limit toxicity.

Tumor suppressor death-associated protein kinase is required for full IL-1ß production.

Interleukin-1ß (IL-1ß) is critical for inflammation and control of infection. The production of IL-1ß depends on expression of pro-IL-1ß and inflammasome component induced by inflammatory stimuli, followed by assembly of inflammasome to generate caspase-1 for cleavage of pro-IL-1ß. Here we show that tumor suppressor death-associated protein kinase (DAPK) deficiency impaired IL-1ß production in macrophages. Generation of tumor necrosis factor-α in macrophages, in contrast, was not affected by DAPK knockout. Two tiers of defects in IL-1ß generation were found in DAPK-deficient macrophages: decreased pro-IL-1ß induction by some stimuli and reduced caspase-1 activation by all inflammatory stimuli examined. With a normal NLRP3 induction in DAPK-deficient macrophages, the diminished caspase-1 generation is attributed to impaired inflammasome assembly. There is a direct binding of DAPK to NLRP3, suggesting an involvement of DAPK in inflammasome formation. We further illustrated that the formation of NLRP3 inflammasome in situ induced by inflammatory signals was impaired by DAPK deficiency. Taken together, our results identify DAPK as a molecule required for full production of IL-1ß and functional assembly of the NLRP3 inflammasome. In addition, DAPK knockout reduced uric acid crystal-triggered peritonitis, suggesting that DAPK may serve as a target in the treatment of IL-1ß-associated autoinflammatory diseases.

Divergent metabolic programmes control two populations of MAIT cells that protect the lung.

Although mucosal-associated invariant T (MAIT) cells provide rapid, innate-like responses, they are not pre-set, and memory-like responses have been described for MAIT cells following infections. The importance of metabolism for controlling these responses, however, is unknown. Here, following pulmonary immunization with a Salmonella vaccine strain, mouse MAIT cells expanded as separate CD127-Klrg1+ and CD127+Klrg1- antigen-adapted populations that differed in terms of their transcriptome, function and localization in lung tissue. These populations remained altered from steady state for months as stable, separate MAIT cell lineages with enhanced effector programmes and divergent metabolism. CD127+ MAIT cells engaged in an energetic, mitochondrial metabolic programme, which was critical for their maintenance and IL-17A synthesis. This programme was supported by high fatty acid uptake and mitochondrial oxidation and relied on highly polarized mitochondria and autophagy. After vaccination, CD127+ MAIT cells protected mice against Streptococcus pneumoniae infection. In contrast, Klrg1+ MAIT cells had dormant but ready-to-respond mitochondria and depended instead on Hif1a-driven glycolysis to survive and produce IFN-γ. They responded antigen independently and participated in protection from influenza virus. These metabolic dependencies may enable tuning of memory-like MAIT cell responses for vaccination and immunotherapies.

Epithelial HVEM maintains intraepithelial T cell survival and contributes to host protection.

Intraepithelial T cells (IETs) are in close contact with intestinal epithelial cells and the underlying basement membrane, and they detect invasive pathogens. How intestinal epithelial cells and basement membrane influence IET survival and function, at steady state or after infection, is unclear. The herpes virus entry mediator (HVEM), a member of the TNF receptor superfamily, is constitutively expressed by intestinal epithelial cells and is important for protection from pathogenic bacteria. Here, we showed that at steady-state LIGHT, an HVEM ligand, binding to epithelial HVEM promoted the survival of small intestine IETs. RNA-seq and addition of HVEM ligands to epithelial organoids indicated that HVEM increased epithelial synthesis of basement membrane proteins, including collagen IV, which bound to ß1 integrins expressed by IETs. Therefore, we proposed that IET survival depended on ß1 integrin binding to collagen IV and showed that ß1 integrin-collagen IV interactions supported IET survival in vitro. Moreover, the absence of ß1 integrin expression by T lymphocytes decreased TCR αß+ IETs in vivo. Intravital microscopy showed that the patrolling movement of IETs was reduced without epithelial HVEM. As likely consequences of decreased number and movement, protective responses to Salmonella enterica were reduced in mice lacking either epithelial HVEM, HVEM ligands, or ß1 integrins. Therefore, IETs, at steady state and after infection, depended on HVEM expressed by epithelial cells for the synthesis of collagen IV by epithelial cells. Collagen IV engaged ß1 integrins on IETs that were important for their maintenance and for their protective function in mucosal immunity.

CD4+-mediated colitis in mice is independent of the GPR183 and GPR18 pathways.

Colitis is characterized by an exacerbated intestinal immune response, but the genetic and other mechanisms regulating immune activation remain incompletely understood. In order to identify new pathways leading to colitis, we sought to identify genes with increased expression in the colons of patients that also are near loci identified by genome wide association studies (GWAS) associated with IBD risk. One such SNP, rs9557195 was of particular interest because it is within an intron of G-protein-coupled receptor (GPR) 183, known to be important for lymphocyte migration. Furthermore, this SNP is in close proximity to the gene encoding another G-protein coupled receptor, GPR18. Analyzing publicly available datasets, we found transcripts of GPR183 and GPR18 to be increased in colon biopsies from ulcerative colitis and Crohn's disease patients, and GPR183 was even more increased in patients resistant to TNF treatment. Expression of both genes also was increased in mouse models of colitis. Therefore, our aim was to understand if increased expression of these GPRs in the intestine is related to disease severity in colitis models. Here we investigated the role of these receptors in the T cell transfer model and the dextran sulfate sodium model. In the T cell transfer model, GPR183 expression on donor T cells, as well as on other cell types in the Rag-/- recipients, was not essential for severe colitis induction. Furthermore, deficiency in Rag-/- mice for the enzyme that synthesizes a cholesterol metabolite that is a major ligand for GPR183 also did not affect disease. Similarly, lack of GPR18 expression in T cells or other cell types did not affect colitis pathogenesis in the T cell transfer or in the dextran sulfate sodium model. Therefore, despite increased expression of transcripts for these genes in the intestine during inflammation in humans and mice, they are not required for disease severity in mouse models of colitis induced by chemical injury or T cell cytokines, perhaps due to redundancy in mechanisms important for homing and survival of lymphocytes to the inflamed intestine.

Transcriptomic Analysis and C-Terminal Epitope Tagging Reveal Differential Processing and Signaling of Endogenous TLR3 and TLR7.

Toll-like receptor (TLR) signaling is critical for defense against pathogenic infection, as well as for modulating tissue development. Activation of different TLRs triggers common inflammatory responses such as cytokine induction. Here, we reveal differential impacts of TLR3 and TLR7 signaling on transcriptomic profiles in bone marrow-derived macrophages (BMDMs). Apart from self-regulation, TLR3, but not TLR7, induced expression of other TLRs, suggesting that TLR3 activation globally enhances innate immunity. Moreover, we observed diverse influences of TLR3 and TLR7 signaling on genes involved in methylation, caspase and autophagy pathways. We compared endogenous TLR3 and TLR7 by using CRISPR/Cas9 technology to knock in a dual Myc-HA tag at the 3' ends of mouse Tlr3 and Tlr7. Using anti-HA antibodies to detect endogenous tagged TLR3 and TLR7, we found that both TLRs display differential tissue expression and posttranslational modifications. C-terminal tagging did not impair TLR3 activity. However, it disrupted the interaction between TLR7 and myeloid differentiation primary response 88 (MYD88), the Tir domain-containing adaptor of TLR7, which blocked its downstream signaling necessary to trigger cytokine and chemokine expression. Our study demonstrates different properties for TLR3 and TLR7, and also provides useful mouse models for further investigation of these two RNA-sensing TLRs.

HVEM structures and mutants reveal distinct functions of binding to LIGHT and BTLA/CD160.

HVEM is a TNF (tumor necrosis factor) receptor contributing to a broad range of immune functions involving diverse cell types. It interacts with a TNF ligand, LIGHT, and immunoglobulin (Ig) superfamily members BTLA and CD160. Assessing the functional impact of HVEM binding to specific ligands in different settings has been complicated by the multiple interactions of HVEM and HVEM binding partners. To dissect the molecular basis for multiple functions, we determined crystal structures that reveal the distinct HVEM surfaces that engage LIGHT or BTLA/CD160, including the human HVEM-LIGHT-CD160 ternary complex, with HVEM interacting simultaneously with both binding partners. Based on these structures, we generated mouse HVEM mutants that selectively recognized either the TNF or Ig ligands in vitro. Knockin mice expressing these muteins maintain expression of all the proteins in the HVEM network, yet they demonstrate selective functions for LIGHT in the clearance of bacteria in the intestine and for the Ig ligands in the amelioration of liver inflammation.

Tumor suppressor death-associated protein kinase 1 inhibits necroptosis by p38 MAPK activation.

Death-associated protein kinase 1 (DAPK1, DAPk, DAPK) is known for its involvement in apoptosis and autophagy-associated cell death. Here, we identified an unexpected function of DAPK1 in suppressing necroptosis. DAPK1-deficiency renders macrophages and dendritic cells susceptible to necroptotic death. We also observed an inhibitory role for DAPK1 in necroptosis in HT-29 cells, since knockdown or knockout of DAPK1 in such cells increased their sensitivity to necroptosis. Increased necroptosis was associated with enhanced formation of the RIPK1-RIPK3-MLKL complex in these DAPK1-deficient cells. We further found that DAPK1-deficiency led to decreased MAPK activated kinase 2 (MK2) activation and reduced RIPK1 S321 phosphorylation, with this latter representing a critical step controlling necrosome formation. Most TNF signaling pathways, including ERK, JNK, and AKT, were not regulated by DAPK. In contrast, DAPK bound p38 MAPK and selectively promoted p38 MAPK activation, resulting in enhanced MK2 phosphorylation. Our results reveal a novel role for DAPK1 in inhibiting necroptosis and illustrate an unexpected selectivity for DAPK1 in promoting p38 MAPK-MK2 activation. Importantly, our study suggests that modulation of necroptosis and p38/MK2-mediated inflammation may be achieved by targeting DAPK1.

Tumour suppressor death-associated protein kinase targets cytoplasmic HIF-1α for Th17 suppression.

Death-associated protein kinase (DAPK) is a tumour suppressor. Here we show that DAPK also inhibits T helper 17 (Th17) and prevents Th17-mediated pathology in a mouse model of autoimmunity. We demonstrate that DAPK specifically downregulates hypoxia-inducible factor 1α (HIF-1α). In contrast to the predominant nuclear localization of HIF-1α in many cell types, HIF-1α is located in both the cytoplasm and nucleus in T cells, allowing for a cytosolic DAPK-HIF-1α interaction. DAPK also binds prolyl hydroxylase domain protein 2 (PHD2) and increases HIF-1α-PHD2 association. DAPK thereby promotes the proline hydroxylation and proteasome degradation of HIF-1α. Consequently, DAPK deficiency leads to excess HIF-1α accumulation, enhanced IL-17 expression and exacerbated experimental autoimmune encephalomyelitis. Additional knockout of HIF-1α restores the normal differentiation of Dapk(-/-) Th17 cells and prevents experimental autoimmune encephalomyelitis development. Our results reveal a mechanism involving DAPK-mediated degradation of cytoplasmic HIF-1α, and suggest that raising DAPK levels could be used for treatment of Th17-associated inflammatory diseases.

Deltex1 antagonizes HIF-1α and sustains the stability of regulatory T cells in vivo.

Application of regulatory T cells (Tregs) in transplantation, autoimmunity and allergy has been extensively explored, but how Foxp3 and Treg stability is regulated in vivo is incompletely understood. Here, we identify a requirement for Deltex1 (DTX1), a contributor to T-cell anergy and Foxp3 protein level maintenance in vivo. Dtx1(-/-) Tregs are as effective as WT Tregs in the inhibition of CD4(+)CD25(-) T-cell activation in vitro. However, the suppressive ability of Dtx1(-/-) Tregs is greatly impaired in vivo. We find that Foxp3 expression is diminished when Dtx1(-/-) Tregs are co-transferred with effector T cells in vivo. DTX1 promotes the degradation of HIF-1α. Knockout of HIF-1α restores the Foxp3 stability and rescues the defective suppressive activity in Dtx1(-/-) Treg cells in vivo. Our results suggest that DTX1 exerts another level of control on Treg stability in vivo by sustaining the expression of Foxp3 protein in Tregs.