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Circadian rhythms are known to regulate immune responses in healthy animals, but it is unclear whether they persist during acute illnesses where clock gene expression is disrupted by systemic inflammation. Here we use a genome-wide approach to investigate circadian gene and metabolite expression in the lungs of endotoxemic mice and find that novel cellular and molecular circadian rhythms are elicited in this setting. The endotoxin-specific circadian programme exhibits unique features, including a divergent group of rhythmic genes and metabolites compared with the basal state and a distinct periodicity and phase distribution. At the cellular level, endotoxin treatment also alters circadian rhythms of leukocyte counts within the lung in a bmal1 -dependent manner, such that granulocytes rather than lymphocytes become the dominant oscillating cell type. Our results show that inflammation produces a complex re-organization of cellular and molecular circadian rhythms that are relevant to early events in lung injury. Whether circadian rhythms occur in settings where clock gene expression is suppressed, such as systemic inflammation, is unclear. Here, the authors examine gene expression and metabolites in the lungs of endotoxemic mice and show that inflammation causes changes in circadian rhythms at the cellular and molecular level.

T cell memory significantly alters the immune response and organ dysfunction induced by the murine cecal ligation and puncture (CLP) model of sepsis. Enhanced T cell memory activation promotes hepatic neutrophilic responses and induces hepatic dysfunction, which is a common complication of human sepsis associated with poor outcomes. We used a novel Immune Educated CLP sepsis mouse model to examine the role of memory T cell cytokine responses in driving innate immunity and organ dysfunction. Through this approach, we found that induced T cell memory prior to CLP led to higher serum levels of IL12, TNF, IL17, and IL1β – all dependent on memory CD8 T cell derived IFNγ following CLP. IFNγ induced activated hepatic IL12 + monocyte-derived dendritic cells. Increased neutrophilic responses occurred in Educated CLP which was found to depend on TNF, and were suppressed by IFNγ. Hepatic dysfunction in response to CLP was worsened by CD4 and CD8 T cell immune memory and prevented by IFNγ and IL17F blockade. These findings were recapitulated in naturalized outbred pet shop mice with natural immune memory to provide translational relevance to our Immune Educated CLP sepsis model. IL17F or IFNγ blockade may represent potential targets for treatment in sepsis with hepatic dysfunction. Graphical Abstract

Background Sepsis is a complex and life-threatening disease process related to a systemic response to severe infection. Due to the challenges of treating patients with sepsis, new therapies are being investigated, including cell-based approaches. Trophoblast stem cells (TSCs) are immune privileged cells with immunomodulatory properties. Thus, we proposed that TSCs may be beneficial in experimental models of sepsis to regulate the immune response and curtail organ injury. Methods Sepsis was induced by experimental models in mice; cecal ligation and puncture (CLP) and lung infection with Streptococcus (S.) pneumoniae. TSCs were isolated from the chorionic villi of human (h) term placentas, and from mouse (m) placentas using anti-CD117 MicroBeads, and were administered intravenously 6 h after CLP or S. pneumoniae infection. We assessed mortality, bacterial clearance, organ injury, inflammatory response, and production of cytokines and chemokines. Results CD117 + hTSCs did not express human leukocyte antigen (HLA) I or II, and were clonogenic and self-renewing. CLP led to severe mortality by 7 days, and administration of either hTSCs or mTSCs resulted in markedly improved survival compared with control cells or vehicle. hTSCs promoted bacterial clearance and decreased organ injury in the liver, kidney, spleen, and bowel. The elevated innate immune response in the peritoneum, predominantly neutrophils, was attenuated by hTSCs. In addition, neutrophil infiltration into the spleen was less in mice receiving hTSCs, which corresponded with reduced plasma pro-inflammatory cytokines and chemokines. When assessing the lung response to S. pneumoniae infection, administration of hTSCs resulted in fewer bacteria in bronchoalveolar lavage fluid (BALF) and lung tissue, and less lung edema and injury. Neutrophils, which were markedly increased in BALF, were diminished and infiltration of neutrophils and macrophages into the lungs was decreased by hTSCs. BALF pro-inflammatory cytokines and chemokines were mitigated by hTSCs to levels of Sham mice, and systemic injury to the liver and spleen was attenuated. Conclusions CD117 + hTSCs are immune privileged cells that when given after the onset of experimental models of infection/sepsis resulted in improved outcomes due to enhanced bacterial clearance, resolving inflammation, and less organ injury. These data support hTSCs as a potential cell-based therapy for sepsis.

BackgroundImmune checkpoint blockade (ICB) response in recurrent/metastatic head and neck squamous cell carcinoma (HNSCC) is limited to 15%–20% of patients and underpinnings of resistance remain undefined.MethodsStarting with an anti-PD1 sensitive murine HNSCC cell line, we generated an isogenic anti-PD1 resistant model. Mass cytometry was used to delineate tumor microenvironments of both sensitive parental murine oral carcinoma (MOC1) and resistant MOC1esc1 tumors. To examine heterogeneity and clonal dynamics of tumor infiltrating lymphocytes (TILs), we applied paired single-cell RNA and TCR sequencing in three HNSCC models.ResultsAnti-PD1 resistant MOC1esc1 line displayed a conserved cell intrinsic immune evasion signature. Immunoprofiling showed distinct baseline tumor microenvironments of MOC1 and MOC1esc1, as well as the remodeling of immune compartments on ICB in MOC1esc1 tumors. Single cell sequencing analysis identified several CD8 +TIL subsets including Tcf7 +Pd1− (naïve/memory-like), Tcf7 +Pd1+ (progenitor), and Tcf7-Pd1+ (differentiated effector). Mapping TCR shared fractions identified that successful anti-PD1 or anti-CTLA4 therapy-induced higher post-treatment T cell lineage transitions.ConclusionsThese data highlight critical aspects of CD8 +TIL heterogeneity and differentiation and suggest facilitation of CD8 +TIL differentiation as a strategy to improve HNSCC ICB response.

Trauma remains one of the leading causes of death worldwide. Traumatic injury disrupts immune system homeostasis and may predispose patients to opportunistic infections and inflammatory complications. Prevention of multiple organ dysfunction syndrome due to septic complications following severe trauma is a challenging problem. Following severe injury, the immune system usually tends toward a pro‐inflammatory phenotype and then changes to a counter‐inflammatory phenotype. This immune system homeostasis is believed to be a protective response based on the balance between the innate and adaptive immune systems. We reported that injury activates inflammasomes and primes Toll‐like receptors. The primed innate immune system is prepared for a rapid and strong antimicrobial immune defense. However, trauma can also develop the “two‐hit” response phenotype. We also reported that injury augments regulatory T cell activity, which can control the “two‐hit” response phenotype in trauma. We discuss the current idea that traumatic injury induces a unique type of innate and adaptive immune response that may be triggered by damage‐associated molecular pattern molecules, which are a combination of endogenous danger signal molecules that include alarmins and pathogen‐associated molecular pattern molecules.

BackgroundIntratumoral viral oncolytic immunotherapy is a promising new approach for the treatment of a variety of solid cancers. CAN-2409 is a replication-deficient adenovirus that delivers herpes simplex virus thymidine kinase to cancer cells, resulting in local conversion of ganciclovir or valacyclovir into a toxic metabolite. This leads to highly immunogenic cell death, followed by a local immune response against a variety of cancer neoantigens and, next, a systemic immune response against the injected tumor and uninjected distant metastases. CAN-2409 treatment has shown promising results in clinical studies in glioblastoma (GBM). Patients with GBM are usually given the corticosteroid dexamethasone to manage edema. Previous work has suggested that concurrent dexamethasone therapy may have a negative effect in patients treated with immune checkpoint inhibitors in patients with GBM. However, the effects of dexamethasone on the efficacy of CAN-2409 treatment have not been explored.MethodsIn vitro experiments included cell viability and neurosphere T-cell killing assays. Effects of dexamethasone on CAN-2409 in vivo were examined using a syngeneic murine GBM model; survival was assessed according to Kaplan-Meier; analyses of tumor-infiltrating lymphocytes were performed with mass cytometry (CyTOF - cytometry by time-of-flight). Data were analyzed using a general linear model, with one-way analysis of variance followed by Dunnett’s multiple comparison test, Kruskal-Wallis test, Dunn’s multiple comparison test or statistical significance analysis of microarrays.ResultsIn a mouse model of GBM, we found that high doses of dexamethasone combined with CAN-2409 led to significantly reduced median survival (29.0 days) compared with CAN-2409 treatment alone (39.5 days). CyTOF analyses of tumor-infiltrating immune cells demonstrated potent immune stimulation induced by CAN-2409 treatment. These effects were diminished when high-dose dexamethasone was used. Functional immune cell characterization suggested increased immune cell exhaustion and tumor promoting profiles after dexamethasone treatment.ConclusionOur data suggest that concurrent high-dose dexamethasone treatment may impair the efficacy of oncolytic viral immunotherapy of GBM, supporting the notion that dexamethasone use should be balanced between symptom control and impact on the therapeutic outcome.

Oligoarticular juvenile idiopathic arthritis (oligo JIA) is the most common form of chronic inflammatory arthritis in children, yet the cause of this disease remains unknown. To understand immune responses in oligo JIA, we immunophenotyped synovial fluid T cells with flow cytometry, bulk RNA-Seq, single-cell RNA-Seq (scRNA-Seq), DNA methylation studies, and Treg suppression assays. In synovial fluid, CD4+, CD8+, and γδ T cells expressed Th1-related markers, whereas Th17 cells were not enriched. Th1 skewing was prominent in CD4+ T cells, including Tregs, and was associated with severe disease. Transcriptomic studies confirmed a Th1 signature in CD4+ T cells from synovial fluid. The regulatory gene expression signature was preserved in Tregs, even those exhibiting Th1 polarization. These Th1-like Tregs maintained Treg-specific methylation patterns and suppressive function, supporting the stability of this Treg population in the joint. Although synovial fluid CD4+ T cells displayed an overall Th1 phenotype, scRNA-Seq uncovered heterogeneous effector and regulatory subpopulations, including IFN-induced Tregs, peripheral helper T cells, and cytotoxic CD4+ T cells. In conclusion, oligo JIA is characterized by Th1 polarization that encompasses Tregs but does not compromise their regulatory identity. Targeting Th1-driven inflammation and augmenting Treg function may represent important therapeutic approaches in oligo JIA.

The authors identify in patients with rheumatoid arthritis a pathogenic subset of CD4+ T cells that augments B cell responses within inflamed tissues. Peripheral helper T cells in rheumatoid arthritis Michael Brenner and colleagues identify a subset of pathogenically activated PD-1 hi CD4-positive T cells in patients with rheumatoid arthritis, and show that it promotes B-cell responses in tertiary lymphoid structures. The cells, which the authors designate as 'peripheral helper' T cells, differ from follicular helper cells in that they lack CXCR5, have altered BCL6 expression, and express chemokine receptors that direct migration to inflamed sites. CD4 + T cells are central mediators of autoimmune pathology; however, defining their key effector functions in specific autoimmune diseases remains challenging. Pathogenic CD4 + T cells within affected tissues may be identified by expression of markers of recent activation 1 . Here we use mass cytometry to analyse activated T cells in joint tissue from patients with rheumatoid arthritis, a chronic immune-mediated arthritis that affects up to 1% of the population 2 . This approach revealed a markedly expanded population of PD-1 hi CXCR5 − CD4 + T cells in synovium of patients with rheumatoid arthritis. However, these cells are not exhausted, despite high PD-1 expression. Rather, using multidimensional cytometry, transcriptomics, and functional assays, we define a population of PD-1 hi CXCR5 − ‘peripheral helper’ T (T PH ) cells that express factors enabling B-cell help, including IL-21, CXCL13, ICOS, and MAF. Like PD-1 hi CXCR5 + T follicular helper cells, T PH cells induce plasma cell differentiation in vitro through IL-21 secretion and SLAMF5 interaction (refs 3 , 4 ). However, global transcriptomics highlight differences between T PH cells and T follicular helper cells, including altered expression of BCL6 and BLIMP1 and unique expression of chemokine receptors that direct migration to inflamed sites, such as CCR2, CX3CR1, and CCR5, in T PH cells. T PH cells appear to be uniquely poised to promote B-cell responses and antibody production within pathologically inflamed non-lymphoid tissues.

Background Acute lung injury (ALI) is a common lung disorder that affects millions of people every year. The infiltration of inflammatory cells into the lungs and death of the alveolar epithelial cells are key factors to trigger a pathological cascade. Trophoblast stem cells (TSCs) are immune privileged, and demonstrate the capability of self-renewal and multipotency with differentiation into three germ layers. We hypothesized that intratracheal transplantation of TSCs may alleviate ALI. Methods ALI was induced by intratracheal delivery of bleomycin (BLM) in mice. After exposure to BLM, pre-labeled TSCs or fibroblasts (FBs) were intratracheally administered into the lungs. Analyses of the lungs were performed for inflammatory infiltrates, cell apoptosis, and engraftment of TSCs. Pro-inflammatory cytokines/chemokines of lung tissue and in bronchoalveolar lavage fluid (BALF) were also assessed. Results The lungs displayed a reduction in cellularity, with decreased CD45 + cells, and less thickening of the alveolar walls in ALI mice that received TSCs compared with ALI mice receiving PBS or FBs. TSCs decreased infiltration of neutrophils and macrophages, and the expression of interleukin (IL) 6, monocyte chemoattractant protein-1 (MCP-1) and keratinocyte-derived chemokine (KC) in the injured lungs. The levels of inflammatory cytokines in BALF, particularly IL-6, were decreased in ALI mice receiving TSCs, compared to ALI mice that received PBS or FBs. TSCs also significantly reduced BLM-induced apoptosis of alveolar epithelial cells in vitro and in vivo. Transplanted TSCs integrated into the alveolar walls and expressed aquaporin 5 and prosurfactant protein C, markers for alveolar epithelial type I and II cells, respectively. Conclusion Intratracheal transplantation of TSCs into the lungs of mice after acute exposure to BLM reduced pulmonary inflammation and cell death. Furthermore, TSCs engrafted into the alveolar walls to form alveolar epithelial type I and II cells. These data support the use of TSCs for the treatment of ALI.