Interactions between immune cell types facilitate the evolution of immune traits.

An essential prerequisite for evolution by natural selection is variation among individuals in traits that affect fitness1. The ability of a system to produce selectable variation, known as evolvability2, thus greatly affects the rate of evolution. The immune system belongs to the fastest evolving components in mammals3, yet the sources of variation in immune traits remain largely unknown4,5. Here, we show that an important determinant of the immune system's evolvability is its organisation into interacting modules represented by different immune cell types. By profiling immune cell variation in bone marrow of 54 genetically diverse mouse strains from the Collaborative Cross6, we found that variation in immune cell frequencies is polygenic and that many associated genes are involved in homeostatic balance through cell-intrinsic functions of proliferation, migration and cell death. However, we also found genes associated with the frequency of a particular cell type, which are expressed in a different cell type, exerting their effect in what we term cyto-trans. Vertebrate evolutionary record shows that genes associated in cyto-trans have faced weaker negative selection, thus increasing the robustness and hence evolvability2,7,8 of the immune system. This phenomenon is similarly observable in human blood. Our findings suggest that interactions between different components of the immune system provide a phenotypic space where mutations can produce variation without much detriment, underscoring the role of modularity in the evolution of complex systems9.

A personalized network framework reveals predictive axis of anti-TNF response across diseases.

Personalized treatment of complex diseases has been mostly predicated on biomarker identification of one drug-disease combination at a time. Here, we use a computational approach termed Disruption Networks to generate a data type, contextualized by cell-centered individual-level networks, that captures biology otherwise overlooked when performing standard statistics. This data type extends beyond the "feature level space", to the "relations space", by quantifying individual-level breaking or rewiring of cross-feature relations. Applying Disruption Networks to dissect high-dimensional blood data, we discover and validate that the RAC1-PAK1 axis is predictive of anti-TNF response in inflammatory bowel disease. Intermediate monocytes, which correlate with the inflammatory state, play a key role in the RAC1-PAK1 responses, supporting their modulation as a therapeutic target. This axis also predicts response in rheumatoid arthritis, validated in three public cohorts. Our findings support blood-based drug response diagnostics across immune-mediated diseases, implicating common mechanisms of non-response.

Alignment of single-cell trajectories by tuMap enables high-resolution quantitative comparison of cancer samples.

Single-cell technologies allow characterization of cancer samples as continuous developmental trajectories. Yet, the obtained temporal resolution cannot be leveraged for a comparative analysis due to the large phenotypic heterogeneity existing between patients. Here, we present the tuMap algorithm that exploits high-dimensional single-cell data of cancer samples exhibiting an underlying developmental structure to align them with the healthy development, yielding the tuMap pseudotime axis that allows their systematic, meaningful comparison. We applied tuMap on single-cell mass cytometry data of acute lymphoblastic and myeloid leukemia to reveal associations between the tuMap pseudotime axis and clinics that outperform cellular assignment into developmental populations. Application of the tuMap algorithm on single-cell RNA sequencing data further identified gene signatures of stem cells residing at the very-early parts of the cancer trajectories. The quantitative framework provided by tuMap allows generation of metrics for cancer patients evaluation.

Autologous Hematological Stem Cell Transplantation for Systemic Sclerosis in Israel.

BACKGROUND: Autologous hematological stem cell transplantation (HSCT) is a novel therapy for systemic sclerosis (SSc) that has been validated in three randomized controlled trials. OBJECTIVES: To report the first Israeli experience with HSCT for progressive SSc and review the current literature. METHODS: Five SSc patients who were evaluated in our department and were treated by HSCT were included. Medical records were evaluated retrospectively. Demographic, clinical, and laboratory data were recorded. Continuous data are presented as the mean ± standard deviation. Categorical variables are presented as frequencies and percentages. RESULTS: Five SSc patients were treated with HSCT. Four patients were adults (mean age 53 ± 12 years) and one was a 12-year-old pediatric patient. All patients were female. HSCT was initiated 1.4 ± 0.8 years after diagnosis. Two patients were RNA POLIII positive, two were anti-topoisomerase 1 positive, and one only antinuclear antibodies positive. All patients had skin and lung involvement. The mean modified Rodnan Skin Score was 29 ± 4.7 before HSCT, which improved to 10.4 ± 9.6 after HSCT. The forced vital capacity improved from 68 ± 13% to 90 ± 28%. Diffusing capacity of the lungs for carbon monoxide increased by 6%. Among severe adverse events were cyclophosphamide-related congestive heart failure, antithymocyte globulin-related capillary leak syndrome, and scleroderma renal crisis. All symptoms completely resolved with treatment without sequela. No treatment related mortality was recorded. CONCLUSIONS: HSCT is an important step in the treatment of progressive SSc in Israel. Careful patient selection reduces treatment related morbidity and mortality.

Pregnancy-Induced Alterations in NK Cell Phenotype and Function.

Pregnant women are particularly susceptible to complications of influenza A virus infection, which may result from pregnancy-induced changes in the function of immune cells, including natural killer (NK) cells. To better understand NK cell function during pregnancy, we assessed the ability of the two main subsets of NK cells, CD56dim, and CD56bright NK cells, to respond to influenza-virus infected cells and tumor cells. During pregnancy, CD56dim and CD56bright NK cells displayed enhanced functional responses to both infected and tumor cells, with increased expression of degranulation markers and elevated frequency of NK cells producing IFN-γ. To better understand the mechanisms driving this enhanced function, we profiled CD56dim and CD56bright NK cells from pregnant and non-pregnant women using mass cytometry. NK cells from pregnant women displayed significantly increased expression of several functional and activation markers such as CD38 on both subsets and NKp46 on CD56dim NK cells. NK cells also displayed diminished expression of the chemokine receptor CXCR3 during pregnancy. Overall, these data demonstrate that functional and phenotypic shifts occur in NK cells during pregnancy that can influence the magnitude of the immune response to both infections and tumors.

Infliximab-Tumor Necrosis Factor Complexes Elicit Formation of Anti-Drug Antibodies.

BACKGROUND & AIMS: Some patients develop anti-drug antibodies (ADAs), which reduce the efficacy of infliximab, a monoclonal antibody against tumor necrosis factor (TNF), in the treatment of immune-mediated diseases, including inflammatory bowel diseases. ADAs arise inconsistently, and it is not clear what factors determine their formation. We investigated features of the immune system, the infliximab antibody, and its complex with TNF that might contribute to ADA generation. METHODS: C57BL/6 mice were given injections of infliximab and recombinant human TNF or infliximab F(ab')2 fragments. Blood samples were collected every 2-3 days for 2 weeks and weekly thereafter for up to 6 weeks; infliximab-TNF complexes and ADAs were measured by enzyme-linked immunosorbent assay (ELISA). Intestinal biopsy and blood samples were obtained from patients having endoscopy who had received infliximab therapy for inflammatory bowel diseases; infliximab-TNF complexes were measured with ELISA. Infliximab-specific plasma cells were detected in patient tissue samples by using mass cytometry. We studied activation of innate immune cells in peripheral blood mononuclear cells (PBMCs) from healthy donors incubated with infliximab or infliximab-TNF complexes; toll-like receptors (TLRs) were blocked with antibodies, endocytosis was blocked with the inhibitor PitStop2, and cytokine expression was measured by real-time polymerase chain reaction and ELISAs. Uptake of infliximab and infliximab-TNF complexes by THP-1 cells was measured with confocal microscopy. RESULTS: Mice given increasing doses of infliximab produced increasing levels of ADAs. Blood samples from mice given injections of human TNF and infliximab contained infliximab-TNF complexes; complex formation was associated with ADA formation with an area under the curve of 0.944 (95% confidence interval, 0.851-1.000; P = .003). Intestinal tissues from patients, but not blood samples, contained infliximab-TNF complexes and infliximab-specific plasma cells. Incubation of PBMCs with infliximab-TNF complexes resulted in a 4.74-fold increase in level of interleukin (IL) 1ß (IL1B) messenger RNA (P for comparison = .005), increased IL1B protein secretion, and a 2.69-fold increase in the expression of TNF messenger RNA (P for comparison = 0.013) compared with control PBMCs. Infliximab reduced only IL1B and TNF expression. Antibodies against TLR2 or TLR4 did not block the increases in IL1B or TNF expression, but endocytosis was required. THP-1 cells endocytosed higher levels of infliximab-TNF complexes than infliximab alone. CONCLUSIONS: In mice, we found ADA formation to increase with dose of infliximab given and concentration of infliximab-TNF complexes detected in blood. Based on studies of human intestinal tissues and blood samples, we propose that infliximab-TNF complexes formed in the intestine are endocytosed by and activate innate immune cells, which increase expression of IL1B and TNF and production of antibodies against the drug complex. It is therefore important to optimize the infliximab dose to a level that is effective but does not activate an innate immune response against the drug-TNF complex.

A clinically meaningful metric of immune age derived from high-dimensional longitudinal monitoring.

Immune responses generally decline with age. However, the dynamics of this process at the individual level have not been characterized, hindering quantification of an individual's immune age. Here, we use multiple 'omics' technologies to capture population- and individual-level changes in the human immune system of 135 healthy adult individuals of different ages sampled longitudinally over a nine-year period. We observed high inter-individual variability in the rates of change of cellular frequencies that was dictated by their baseline values, allowing identification of steady-state levels toward which a cell subset converged and the ordered convergence of multiple cell subsets toward an older adult homeostasis. These data form a high-dimensional trajectory of immune aging (IMM-AGE) that describes a person's immune status better than chronological age. We show that the IMM-AGE score predicted all-cause mortality beyond well-established risk factors in the Framingham Heart Study, establishing its potential use in clinics for identification of patients at risk.

Cell-centred meta-analysis reveals baseline predictors of anti-TNFα non-response in biopsy and blood of patients with IBD.

OBJECTIVE: Although anti-tumour necrosis factor alpha (anti-TNFα) therapies represent a major breakthrough in IBD therapy, their cost-benefit ratio is hampered by an overall 30% non-response rate, adverse side effects and high costs. Thus, finding predictive biomarkers of non-response prior to commencing anti-TNFα therapy is of high value. DESIGN: We analysed publicly available whole-genome expression profiles of colon biopsies obtained from multiple cohorts of patients with IBD using a combined computational deconvolution-meta-analysis paradigm which allows to estimate immune cell contribution to the measured expression and capture differential regulatory programmes otherwise masked due to variation in cellular composition. Insights from this in silico approach were experimentally validated in biopsies and blood samples of three independent test cohorts. RESULTS: We found the proportion of plasma cells as a robust pretreatment biomarker of non-response to therapy, which we validated in two independent cohorts of immune-stained colon biopsies, where a plasma cellular score from inflamed biopsies was predictive of non-response with an area under the curve (AUC) of 82%. Meta-analysis of the cell proportion-adjusted gene expression data suggested that an increase in inflammatory macrophages in anti-TNFα non-responding individuals is associated with the upregulation of the triggering receptor expressed on myeloid cells 1 (TREM-1) and chemokine receptor type 2 (CCR2)-chemokine ligand 7 (CCL7) -axes. Blood gene expression analysis of an independent cohort, identified TREM-1 downregulation in non-responders at baseline, which was predictive of response with an AUC of 94%. CONCLUSIONS: Our study proposes two clinically feasible assays, one in biopsy and one in blood, for predicting non-response to anti-TNFα therapy prior to initiation of treatment. Moreover, it suggests that mechanism-driven novel drugs for non-responders should be developed.

Found In Translation: a machine learning model for mouse-to-human inference.

Cross-species differences form barriers to translational research that ultimately hinder the success of clinical trials, yet knowledge of species differences has yet to be systematically incorporated in the interpretation of animal models. Here we present Found In Translation (FIT; http://www.mouse2man.org ), a statistical methodology that leverages public gene expression data to extrapolate the results of a new mouse experiment to expression changes in the equivalent human condition. We applied FIT to data from mouse models of 28 different human diseases and identified experimental conditions in which FIT predictions outperformed direct cross-species extrapolation from mouse results, increasing the overlap of differentially expressed genes by 20-50%. FIT predicted novel disease-associated genes, an example of which we validated experimentally. FIT highlights signals that may otherwise be missed and reduces false leads, with no experimental cost.

Regulation of S100A8 Stability by RNF5 in Intestinal Epithelial Cells Determines Intestinal Inflammation and Severity of Colitis.

Inflammatory bowel disease (IBD) is prevalent, but the mechanisms underlying disease development remain elusive. We identify a role for the E3 ubiquitin ligase RNF5 in IBD. Intestinal epithelial cells (IECs) express a high level of RNF5, while the colon of Rnf5-/- mice exhibits activated dendritic cells and intrinsic inflammation. Rnf5-/- mice exhibit severe acute colitis following dextran sodium sulfate (DSS) treatment. S100A8 is identified as an RNF5 substrate, resulting in S100A8 ubiquitination and proteasomal-dependent degradation that is attenuated upon inflammatory stimuli. Loss of RNF5 from IECs leads to enhanced S100A8 secretion, which induces mucosal CD4+ T cells, resulting in Th1 pro-inflammatory responses. Administration of S100A8-neutralizing antibodies to DSS-treated Rnf5-/- mice attenuates acute colitis development and increases survival. An inverse correlation between RNF5 and S100A8 protein expression in IECs of IBD patients coincides with disease severity. Collectively, RNF5-mediated regulation of S100A8 stability in IECs is required for the maintenance of intestinal homeostasis.

Immune-centric network of cytokines and cells in disease context identified by computational mining of PubMed.

Cytokines are signaling molecules secreted and sensed by immune and other cell types, enabling dynamic intercellular communication. Although a vast amount of data on these interactions exists, this information is not compiled, integrated or easily searchable. Here we report immuneXpresso, a text-mining engine that structures and standardizes knowledge of immune intercellular communication. We applied immuneXpresso to PubMed to identify relationships between 340 cell types and 140 cytokines across thousands of diseases. The method is able to distinguish between incoming and outgoing interactions, and it includes the effect of the interaction and the cellular function involved. These factors are assigned a confidence score and linked to the disease. By leveraging the breadth of this network, we predicted and experimentally verified previously unappreciated cell-cytokine interactions. We also built a global immune-centric view of diseases and used it to predict cytokine-disease associations. This standardized knowledgebase (http://www.immunexpresso.org) opens up new directions for interpretation of immune data and model-driven systems immunology.

Activation of the reward system boosts innate and adaptive immunity.

Positive expectations contribute to the clinical benefits of the placebo effect. Such positive expectations are mediated by the brain's reward system; however, it remains unknown whether and how reward system activation affects the body's physiology and, specifically, immunity. Here we show that activation of the ventral tegmental area (VTA), a key component of the reward system, strengthens immunological host defense. We used 'designer receptors exclusively activated by designer drugs' (DREADDs) to directly activate dopaminergic neurons in the mouse VTA and characterized the subsequent immune response after exposure to bacteria (Escherichia coli), using time-of-flight mass cytometry (CyTOF) and functional assays. We found an increase in innate and adaptive immune responses that were manifested by enhanced antibacterial activity of monocytes and macrophages, reduced in vivo bacterial load and a heightened T cell response in the mouse model of delayed-type hypersensitivity. By chemically ablating the sympathetic nervous system (SNS), we showed that the reward system's effects on immunity are, at least partly, mediated by the SNS. Thus, our findings establish a causal relationship between the activity of the VTA and the immune response to bacterial infection.

STAT3 regulates uterine epithelial remodeling and epithelial-stromal crosstalk during implantation.

Embryo implantation is regulated by a variety of endometrial factors, including cytokines, growth factors, and transcription factors. Earlier studies identified the leukemia inhibitory factor (LIF), a cytokine produced by uterine glands, as an essential regulator of implantation. LIF, acting via its cell surface receptor, activates the signal transducer and activator of transcription 3 (STAT3) in the uterine epithelial cells. However, the precise mechanism via which activated STAT3 promotes uterine function during implantation remains unknown. To identify the molecular pathways regulated by STAT3, we created SW(d/d) mice in which Stat3 gene is conditionally inactivated in uterine epithelium. The SW(d/d) mice are infertile due to a lack of embryo attachment to the uterine luminal epithelium and consequent implantation failure. Gene expression profiling of uterine epithelial cells of SW(d/d) mice revealed dysregulated expression of specific components of junctional complexes, including E-cadherin, α- and ß-catenin, and several claudins, which critically regulate epithelial junctional integrity and embryo attachment. In addition, uteri of SW(d/d) mice exhibited markedly reduced stromal proliferation and differentiation, indicating that epithelial STAT3 controls stromal function via a paracrine mechanism. The stromal defect arose from a drastic reduction in the production of several members of the epidermal growth factor family in luminal epithelium of SW(d/d) uteri and the resulting lack of activation of epidermal growth factor receptor signaling and mitotic activity in the stromal cells. Collectively, our results uncovered an intricate molecular network operating downstream of STAT3 that regulates uterine epithelial junctional reorganization, and stromal proliferation, and differentiation, which are critical determinants of successful implantation.

Sequential phosphorylation of SLP-76 at tyrosine 173 is required for activation of T and mast cells.

Cooperatively assembled signalling complexes, nucleated by adaptor proteins, integrate information from surface receptors to determine cellular outcomes. In T and mast cells, antigen receptor signalling is nucleated by three adaptors: SLP-76, Gads and LAT. Three well-characterized SLP-76 tyrosine phosphorylation sites recruit key components, including a Tec-family tyrosine kinase, Itk. We identified a fourth, evolutionarily conserved SLP-76 phosphorylation site, Y173, which was phosphorylated upon T-cell receptor stimulation in primary murine and Jurkat T cells. Y173 was required for antigen receptor-induced phosphorylation of phospholipase C-γ1 (PLC-γ1) in both T and mast cells, and for consequent downstream events, including activation of the IL-2 promoter in T cells, and degranulation and IL-6 production in mast cells. In intact cells, Y173 phosphorylation depended on three, ZAP-70-targeted tyrosines at the N-terminus of SLP-76 that recruit and activate Itk, a kinase that selectively phosphorylated Y173 in vitro. These data suggest a sequential mechanism whereby ZAP-70-dependent priming of SLP-76 at three N-terminal sites triggers reciprocal regulatory interactions between Itk and SLP-76, which are ultimately required to couple active Itk to its substrate, PLC-γ1.