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Competing Interests: Declaration of interests D.C. is a co-inventor on two patents related to predicting cancer immunotherapy response. The first patent (US11230599/EP4226944A3), filed by MSKCC, covers the use of tumor mutational burden for this purpose and is licensed to Personal Genome Diagnostics (PGDx). The second patent (US20240282410A1), filed jointly by Cleveland Clinic and MSKCC, describes a multi-modal machine learning model for predicting immunotherapy response and is licensed to Tempus. K.M.-J. is affiliated with Yale University.

Hematopoiesis is a highly dynamical and stochastic process, challenging our understanding of homeostasis. Clinical studies of leukemia or neutropenic patients revealed that multiple blood cell types fluctuate spontaneously with large yet regular oscillations of their frequencies. Yet the stability of hematopoiesis in healthy individuals remains understudied. Here we report on both cross-sectional and longitudinal studies of dozens of healthy mice, through high-dimensional mass and spectral cytometry, to understand hematopoiesis at homeostasis. We found that all cell types in the bone marrow, blood, and spleen exhibit large variations of frequency (e.g., with coefficients of variation larger than 1). While the frequencies of individual cell type fluctuate, there existed extensive and robust correlations/anti-correlations between cell types, exemplified by the pronounced anti-correlation between blood neutrophils and B cells. Through longitudinal study of the blood content of healthy mice, we found that leukocyte fluctuations are ergodic yet subject to chaotic behaviors characterized by a broad spectrum of characteristic timescales. We then built a minimal mathematical model to capture these dynamical features of hematopoiesis (fluctuations, correlations, and chaos) and explain how the accumulation of B cells (e.g. during lymphoma development) would transition the blood cell dynamics from chaos to oscillations (as observed clinically). Finally, we demonstrated the ubiquity and consistency of the correlated fluctuations in hematopoiesis by comparing mouse cohorts of different genetic backgrounds and ages. To conclude, we discuss how study of hematopoiesis must factor in the newfound chaotic dynamics at homeostasis, towards better modeling the responses to perturbations.

Immune responses against cancer are inherently stochastic, with small numbers of individual T cells within a larger ensemble of lymphocytes initiating the molecular cascades that lead to tumor cytotoxicity. A potential source of this intra-tumor variability is the differential ability of immune cells to respond to tumor cells. Classical microwell co-cultures of T cells and tumor cells are inadequate for reliably culturing and analyzing low cell numbers needed to probe this variability, and have failed in recapitulating the heterogeneous small domains observed in tumors. Here we leverage a membrane displacement trap array technology that overcomes limitations of conventional microwell plates for immunodynamic studies. The microfluidic platform supports on-demand formation of dense nanowell cultures under continuous perfusion reflecting the tumor microenvironment, with real-time monitoring of T cell proliferation and activation within each nanowell. The system enables selective ejection of cells for profiling by fluorescence activated cell sorting, allowing observed on-chip variability in immune response to be correlated with off-chip quantification of T cell activation. The technology offers new potential for probing the molecular origins of T cell heterogeneity and identifying specific cell phenotypes responsible for initiating and propagating immune cascades within tumors. Insight Box Variability in T cell activation plays a critical role in the immune response against cancer. New tools are needed to unravel the mechanisms that drive successful anti-tumor immune response, and to support the development of novel immunotherapies utilizing rare T cell phenotypes that promote effective immune surveillance. To this end, we present a microfluidic cell culture platform capable of probing differential T cell activation in an array of nanoliter-scale wells coupled with off-chip cell analysis, enabling a high resolution view of variable immune response within tumor / T cell co-cultures containing cell ensembles orders of magnitude smaller than conventional well plate studies., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Chimeric antigen receptor T cells (CART) have demonstrated curative potential for hematological malignancies, but the optimal manufacturing has not yet been determined and may differ across products. The first step, T cell selection, removes contaminating cell types that can potentially suppress T cell expansion and transduction. While positive selection of CD4/CD8 T cells after leukapheresis is often used in clinical trials, it may modulate signaling cascades downstream of these co-receptors; indeed, the addition of a CD4/CD8-positive selection step altered CD22 CART potency and toxicity in patients. While negative selection may avoid this drawback, it is virtually absent from good manufacturing practices. Here, we performed both CD4/CD8-positive and -negative clinical scale selections of mononuclear cell apheresis products and generated CD22 CARTs per our ongoing clinical trial (NCT02315612NCT02315612). While the selection process did not yield differences in CART expansion or transduction, positively selected CART exhibited a significantly higher in vitro interferon-γ and IL-2 secretion but a lower in vitro tumor killing rate. Notably, though, CD22 CART generated from both selection protocols efficiently eradicated leukemia in NSG mice, with negatively selected cells exhibiting a significant enrichment in γδ CD22 CART. Thus, our study demonstrates the importance of the initial T cell selection process in clinical CART manufacturing., Competing Interests: V.P. is an employee of STEMCELL Technologies, Inc., (© 2023.)

Checkpoint blockade revolutionized cancer therapy, but we still lack a quantitative, mechanistic understanding of how inhibitory receptors affect diverse signaling pathways. To address this issue, we developed and applied a fluorescent intracellular live multiplex signal transduction activity reporter (FILMSTAR) system to analyze PD-1-induced suppressive effects. These studies identified pathways triggered solely by TCR or requiring both TCR and CD28 inputs. Using presenting cells differing in PD-L1 and CD80 expression while displaying TCR ligands of distinct potency, we found that PD-1-mediated inhibition primarily targets TCR-linked signals in a manner highly sensitive to peptide ligand quality. These findings help resolve discrepancies in existing data about the site(s) of PD-1 inhibition in T cells while emphasizing the importance of neoantigen potency in controlling the effects of checkpoint therapy., (© 2023 Chan et al.)

The T cell antigen receptor (TCR) contains ten immunoreceptor tyrosine-based activation motif (ITAM) signaling sequences distributed within six CD3 subunits; however, the reason for such structural complexity and multiplicity is unclear. Here we evaluated the effect of inactivating the three CD3ζ chain ITAMs on TCR signaling and T cell effector responses using a conditional 'switch' mouse model. Unexpectedly, we found that T cells expressing TCRs containing inactivated (non-signaling) CD3ζ ITAMs (6F-CD3ζ) exhibited reduced ability to discriminate between low- and high-affinity ligands, resulting in enhanced signaling and cytokine responses to low-affinity ligands because of a previously undetected inhibitory function of CD3ζ ITAMs. Also, 6F-CD3ζ TCRs were refractory to antagonism, as predicted by a new in silico adaptive kinetic proofreading model that revises the role of ITAM multiplicity in TCR signaling. Finally, T cells expressing 6F-CD3ζ displayed enhanced cytolytic activity against solid tumors expressing low-affinity ligands, identifying a new counterintuitive approach to TCR-mediated cancer immunotherapy., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Cytotoxic T lymphocytes (CTLs) fight intracellular pathogens and cancer by identifying and destroying infected or transformed target cells 1 . To kill, CTLs form a specialized cytotoxic immune synapse (IS) with a target of interest and then release toxic perforin and granzymes into the interface to elicit programmed cell death 2-5 . The IS then dissolves, enabling CTLs to search for additional prey and professional phagocytes to clear the corpse 6 . While the mechanisms governing IS assembly have been studied extensively, far less is known about target cell release. Here, we applied time-lapse imaging to explore the basis for IS dissolution and found that it occurred concomitantly with the cytoskeletal contraction of apoptotic targets. Genetic and pharmacological perturbation of this contraction response indicated that it was both necessary and sufficient for CTL dissociation. We also found that mechanical amplification of apoptotic contractility promoted faster CTL detachment and serial killing. Collectively, these results establish a biophysical basis for IS dissolution and highlight the importance of mechanosensory feedback in the regulation of cell-cell interactions., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

CD19 chimeric antigen receptor T-cell therapy (CD19-CAR) has changed the treatment landscape and outcomes for patients with pre-B-cell acute lymphoblastic leukemia (B-ALL). Unfortunately, primary nonresponse (PNR), sustained CD19+ disease, and concurrent expansion of CD19-CAR occur in 20% of the patients and is associated with adverse outcomes. Although some failures may be attributable to CD19 loss, mechanisms of CD19-independent, leukemia-intrinsic resistance to CD19-CAR remain poorly understood. We hypothesize that PNR leukemias are distinct compared with primary sensitive (PS) leukemias and that these differences are present before treatment. We used a multiomic approach to investigate this in 14 patients (7 with PNR and 7 with PS) enrolled in the PLAT-02 trial at Seattle Children's Hospital. Long-read PacBio sequencing helped identify 1 PNR in which 47% of CD19 transcripts had exon 2 skipping, but other samples lacked CD19 transcript abnormalities. Epigenetic profiling discovered DNA hypermethylation at genes targeted by polycomb repressive complex 2 (PRC2) in embryonic stem cells. Similarly, assays of transposase-accessible chromatin-sequencing revealed reduced accessibility at these PRC2 target genes, with a gain in accessibility of regions characteristic of hematopoietic stem cells and multilineage progenitors in PNR. Single-cell RNA sequencing and cytometry by time of flight analyses identified leukemic subpopulations expressing multilineage markers and decreased antigen presentation in PNR. We thus describe the association of a stem cell epigenome with primary resistance to CD19-CAR therapy. Future trials incorporating these biomarkers, with the addition of multispecific CAR T cells targeting against leukemic stem cell or myeloid antigens, and/or combined epigenetic therapy to disrupt this distinct stem cell epigenome may improve outcomes of patients with B-ALL., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Designer lymphocytes expand the dynamic range of possibilities for treating disease.

Data clustering plays a significant role in biomedical sciences, particularly in single-cell data analysis. Researchers use clustering algorithms to group individual cells into populations that can be evaluated across different levels of disease progression, drug response, and other clinical statuses. In many cases, multiple sets of clusters must be generated to assess varying levels of cluster specificity. For example, there are many subtypes of leukocytes (e.g. T cells), whose individual preponderance and phenotype must be assessed for statistical/functional significance. In this report, we introduce a novel hierarchical density clustering algorithm (HAL-x) that uses supervised linkage methods to build a cluster hierarchy on raw single-cell data. With this new approach, HAL-x can quickly predict multiple sets of labels for immense datasets, achieving a considerable improvement in computational efficiency on large datasets compared to existing methods. We also show that cell clusters generated by HAL-x yield near-perfect F1-scores when classifying different clinical statuses based on single-cell profiles. Our hierarchical density clustering algorithm achieves high accuracy in single cell classification in a scalable, tunable and rapid manner., Competing Interests: The authors have declared that no competing interests exist.

Remission durability following single-antigen targeted chimeric antigen receptor (CAR) T-cells is limited by antigen modulation, which may be overcome with combinatorial targeting. Building upon our experiences targeting CD19 and CD22 in B-cell acute lymphoblastic leukemia (B-ALL), we report on our phase 1 dose-escalation study of a novel murine stem cell virus (MSCV)-CD19/CD22-4-1BB bivalent CAR T-cell (CD19.22.BBζ) for children and young adults (CAYA) with B-cell malignancies. Primary objectives included toxicity and dose finding. Secondary objectives included response rates and relapse-free survival (RFS). Biologic correlatives included laboratory investigations, CAR T-cell expansion and cytokine profiling. Twenty patients, ages 5.4 to 34.6 years, with B-ALL received CD19.22.BBζ. The complete response (CR) rate was 60% (12 of 20) in the full cohort and 71.4% (10 of 14) in CAR-naïve patients. Ten (50%) developed cytokine release syndrome (CRS), with 3 (15%) having ≥ grade 3 CRS and only 1 experiencing neurotoxicity (grade 3). The 6- and 12-month RFS in those achieving CR was 80.8% (95% confidence interval [CI]: 42.4%-94.9%) and 57.7% (95% CI: 22.1%-81.9%), respectively. Limited CAR T-cell expansion and persistence of MSCV-CD19.22.BBζ compared with EF1α-CD22.BBζ prompted laboratory investigations comparing EF1α vs MSCV promoters, which did not reveal major differences. Limited CD22 targeting with CD19.22.BBζ, as evaluated by ex vivo cytokine secretion and leukemia eradication in humanized mice, led to development of a novel bicistronic CD19.28ζ/CD22.BBζ construct with enhanced cytokine production against CD22. With demonstrated safety and efficacy of CD19.22.BBζ in a heavily pretreated CAYA B-ALL cohort, further optimization of combinatorial antigen targeting serves to overcome identified limitations (www.clinicaltrials.gov #NCT03448393)., (© 2022 by The American Society of Hematology.)

Systems immunology lacks a framework with which to derive theoretical understanding from high-dimensional datasets. We combined a robotic platform with machine learning to experimentally measure and theoretically model CD8 + T cell activation. High-dimensional cytokine dynamics could be compressed onto a low-dimensional latent space in an antigen-specific manner (so-called "antigen encoding"). We used antigen encoding to model and reconstruct patterns of T cell immune activation. The model delineated six classes of antigens eliciting distinct T cell responses. We generalized antigen encoding to multiple immune settings, including drug perturbations and activation of chimeric antigen receptor T cells. Such universal antigen encoding for T cell activation may enable further modeling of immune responses and their rational manipulation to optimize immunotherapies.

Cognate antigen signal controls CD8 + T cell priming, expansion size and effector versus memory cell fates, but it is not known if and how it modulates the functional features of memory CD8 + T cells. Here we show that the strength of T cell receptor (TCR) signaling controls the requirement for interleukin-2 (IL-2) signals to form a pool of memory CD8 + T cells that competitively re-expand upon secondary antigen encounter. Combining strong TCR and intact IL-2 signaling during priming synergistically induces genome-wide chromatin accessibility in regions targeting a wide breadth of biological processes, consistent with greater T cell functional fitness. Chromatin accessibility in promoters of genes encoding for stem cell, cell cycle and calcium-related proteins correlates with faster intracellular calcium accumulation, initiation of cell cycle and more robust expansion. High-dimensional flow-cytometry analysis of these T cells also highlights higher diversity of T cell subsets and phenotypes with T cells primed with stronger TCR and IL-2 stimulation than those primed with weaker strengths of TCR and/or IL-2 signals. These results formally show that epitope selection in vaccine design impacts memory CD8 + T cell epigenetic programming and function., (© 2022. The Author(s).)

Interactions between human leukocyte antigen (HLA) molecules on target cells and the inhibitory killer cell immunoglobulin-like receptors (KIRs) and heterodimeric inhibitory receptor CD94-NKG2A on human natural killer (NK) cells shape and program various response capacities. A functionally orthologous system exists in mice, consisting of major histocompatibility complex (MHC) molecules on target cells and the inhibitory Ly49 and CD94-NKG2A receptors on NK cells. Here, we found that the abundance of Src homology 2 domain–containing phosphatase-1 (SHP-1) in NK cells was established by interactions between MHCs and NK cell inhibitory receptors, although phenotypically identical NK cell populations still showed substantial variability in endogenous SHP-1 abundance and NK cell response potential. Human and mouse NK cell populations with high responsiveness had low SHP-1 abundance, and a reduction in SHP-1 abundance in NK cells enhanced their responsiveness. Computational modeling of NK cell activation by membrane-proximal signaling events identified SHP-1 as a negative amplitude regulator, which was validated by single-cell analysis of human NK cell responsiveness. The amount of mRNA and protein varied among responsive NK cells despite their similar chromatin accessibility to that of unresponsive cells, suggesting dynamic regulation of SHP-1 abundance. Low intracellular SHP-1 abundance was a biomarker of responsive NK cells. Together, these data suggest that enhancing NK cell function through the acute loss of SHP-1 abundance or activity may enhance the tumoricidal capacity of NK cells.

The Type I Interferon family of cytokines all act through the same cell surface receptor and induce phosphorylation of the same subset of response regulators of the STAT family. Despite their shared receptor, different Type I Interferons have different functions during immune response to infection. In particular, they differ in the potency of their induced anti-viral and anti-proliferative responses in target cells. It remains not fully understood how these functional differences can arise in a ligand-specific manner both at the level of STAT phosphorylation and the downstream function. We use a minimal computational model of Type I Interferon signaling, focusing on Interferon- α and Interferon- β . We validate the model with quantitative experimental data to identify the key determinants of specificity and functional plasticity in Type I Interferon signaling. We investigate different mechanisms of signal discrimination, and how multiple system components such as binding affinity, receptor expression levels and their variability, receptor internalization, short-term negative feedback by SOCS1 protein, and differential receptor expression play together to ensure ligand specificity on the level of STAT phosphorylation. Based on these results, we propose phenomenological functional mappings from STAT activation to downstream anti-viral and anti-proliferative activity to investigate differential signal processing steps downstream of STAT phosphorylation. We find that the negative feedback by the protein USP18, which enhances differences in signaling between Interferons via ligand-dependent refractoriness, can give rise to functional plasticity in Interferon- α and Interferon- β signaling, and explore other factors that control functional plasticity. Beyond Type I Interferon signaling, our results have a broad applicability to questions of signaling specificity and functional plasticity in signaling systems with multiple ligands acting through a bottleneck of a small number of shared receptors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kirby, Parmar, Fathi, Marwah, Nayak, Cherepanov, MacParland, Feld, Altan-Bonnet and Zilman.)

Cytokines are critical for intercellular communication in human health and disease, but the investigation of cytokine signaling activity has remained challenging due to the short half-lives of cytokines and the complexity/redundancy of cytokine functions. To address these challenges, we developed the Cytokine Signaling Analyzer (CytoSig; https://cytosig.ccr.cancer.gov/ ), providing both a database of target genes modulated by cytokines and a predictive model of cytokine signaling cascades from transcriptomic profiles. We collected 20,591 transcriptome profiles for human cytokine, chemokine and growth factor responses. This atlas of transcriptional patterns induced by cytokines enabled the reliable prediction of signaling activities in distinct cell populations in infectious diseases, chronic inflammation and cancer using bulk and single-cell transcriptomic data. CytoSig revealed previously unidentified roles of many cytokines, such as BMP6 as an anti-inflammatory factor, and identified candidate therapeutic targets in human inflammatory diseases, such as CXCL8 for severe coronavirus disease 2019., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

A fraction of mature T cells can be activated by peripheral self-antigens, potentially eliciting host autoimmunity. We investigated homeostatic control of self-activated T cells within unperturbed tissue environments by combining high-resolution multiplexed and volumetric imaging with computational modeling. In lymph nodes, self-activated T cells produced interleukin (IL)-2, which enhanced local regulatory T cell (Treg) proliferation and inhibitory functionality. The resulting micro-domains reciprocally constrained inputs required for damaging effector responses, including CD28 co-stimulation and IL-2 signaling, constituting a negative feedback circuit. Due to these local constraints, self-activated T cells underwent transient clonal expansion, followed by rapid death ("pruning"). Computational simulations and experimental manipulations revealed the feedback machinery's quantitative limits: modest reductions in Treg micro-domain density or functionality produced non-linear breakdowns in control, enabling self-activated T cells to subvert pruning. This fine-tuned, paracrine feedback process not only enforces immune homeostasis but also establishes a sharp boundary between autoimmune and host-protective T cell responses., Competing Interests: Declaration of interests A.Y.R. is a co-founder of Sonoma Biotherapeutics; he is an SAB member and reports personal fees from Sonoma Biotherapeutics, RAPT Therapeutics, and Vedanta Biosciences and holds an IP licensed to Takeda all outside the submitted work. All other authors declare no competing interests., (Published by Elsevier Inc.)

Adoptive T cell therapy (ACT) using ex vivo-expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate complete regression of certain human cancers. The impact of TIL phenotypes on clinical success of TIL-ACT is currently unclear. Using high-dimensional analysis of human ACT products, we identified a memory-progenitor CD39-negative stem-like phenotype (CD39 - CD69 - ) associated with complete cancer regression and TIL persistence and a terminally differentiated CD39-positive state (CD39 + CD69 + ) associated with poor TIL persistence. Most antitumor neoantigen-reactive TILs were found in the differentiated CD39 + state. However, ACT responders retained a pool of CD39 - stem-like neoantigen-specific TILs that was lacking in ACT nonresponders. Tumor-reactive stem-like TILs were capable of self-renewal, expansion, persistence, and superior antitumor response in vivo. These data suggest that TIL subsets mediating ACT response are distinct from TIL subsets enriched for antitumor reactivity., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

β-Coronaviruses are a family of positive-strand enveloped RNA viruses that includes the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Much is known regarding their cellular entry and replication pathways, but their mode of egress remains uncertain. Using imaging methodologies and virus-specific reporters, we demonstrate that β-coronaviruses utilize lysosomal trafficking for egress rather than the biosynthetic secretory pathway more commonly used by other enveloped viruses. This unconventional egress is regulated by the Arf-like small GTPase Arl8b and can be blocked by the Rab7 GTPase competitive inhibitor CID1067700. Such non-lytic release of β-coronaviruses results in lysosome deacidification, inactivation of lysosomal degradation enzymes, and disruption of antigen presentation pathways. β-Coronavirus-induced exploitation of lysosomal organelles for egress provides insights into the cellular and immunological abnormalities observed in patients and suggests new therapeutic modalities., Competing Interests: Declaration of Interests The authors declare no competing interests., (Published by Elsevier Inc.)

The incidence of thyroid cancer, the most frequent endocrine neoplasia, is rapidly increasing. Significant progress has recently been made in the identification of genetic lesions in thyroid cancer; however, whether inflammation contributes to thyroid cancer progression remains unknown. Using a mouse model of aggressive follicular thyroid cancer (FTC; Thrb PV/PV Pten +/- mice), we aimed to elucidate a cause-effect relationship at the molecular level. The Thrb PV/PV Pten +/- mouse expresses a dominantly negative thyroid hormone receptor β (denoted as PV) and a deletion of a single allele of the Pten gene. These two oncogenic signaling pathways synergistically activate PI3K-AKT signaling to drive cancer progression as in human FTC. At the age of 5-7 weeks, thyroids of Thrb PV/PV Pten +/- mice exhibited extensive hyperplasia accompanied by 77.5-fold infiltration of inflammatory monocytes as compared with normal thyroids. Global gene expression profiling identified altered expression of 2387 genes, among which 1353 were upregulated and 1034 were down-regulated. Further analysis identified markedly elevated expression of inflammation mediators and cytokines such as, Csf1r, Csf1, SPP1, Aif1, IL6, Ccl9, Ccl3, Ccl12 , and Ccr2 genes and decreased expression of Kit, Ephx2, Cd163, IL15, Ccl11 , and Cxcl13 genes. These changes elicited the inflammatory responses in the hyperplastic thyroid of Thrb PV/PV Pten +/- mice, reflecting early events in thyroid carcinogenesis. We next tested whether attenuating the inflammatory responses could mitigate thyroid cancer progression. We treated the mice with an inhibitor of colony-stimulating factor 1 receptor (CSF1R), pexidartinib (PLX-3397; PLX). CSF1R mediates the activity of the cytokine, colony stimulating factor 1 (CSF1), in the production, differentiation, and functions of monocytes and macrophages. Treatment with PLX decreased 94% and 62% of inflammatory monocytes in the thyroid and bone marrow, respectively, versus controls. Further, PLX suppressed the expression of critical cytokine and inflammation-regulating genes such as Csf1r, SPP1 (OPN), Aif1, IL6, Ccl9, Ccl3, Ccl12 , and Ccr2 (25%-80%), resulting in inhibition of 89% tumor cell proliferation, evidenced by Ki-67 immunostaining. These preclinical findings suggest that inflammation occurs in the early stage of thyroid carcinogenesis and plays a critical in cancer progression. Importantly, attenuation of inflammation by inhibitors such as PLX would be beneficial in preventing thyroid cancer., Competing Interests: None., (AJCR Copyright © 2020.)

X-linked immunodeficiency with magnesium defect, EBV infection, and neoplasia (XMEN) disease are caused by deficiency of the magnesium transporter 1 (MAGT1) gene. We studied 23 patients with XMEN, 8 of whom were EBV naive. We observed lymphadenopathy (LAD), cytopenias, liver disease, cavum septum pellucidum (CSP), and increased CD4-CD8-B220-TCRαβ+ T cells (αβDNTs), in addition to the previously described features of an inverted CD4/CD8 ratio, CD4+ T lymphocytopenia, increased B cells, dysgammaglobulinemia, and decreased expression of the natural killer group 2, member D (NKG2D) receptor. EBV-associated B cell malignancies occurred frequently in EBV-infected patients. We studied patients with XMEN and patients with autoimmune lymphoproliferative syndrome (ALPS) by deep immunophenotyping (32 immune markers) using time-of-flight mass cytometry (CyTOF). Our analysis revealed that the abundance of 2 populations of naive B cells (CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4++CD10+CD38+ and CD20+CD27-CD22+IgM+HLA-DR+CXCR5+CXCR4+CD10-CD38-) could differentially classify XMEN, ALPS, and healthy individuals. We also performed glycoproteomics analysis on T lymphocytes and show that XMEN disease is a congenital disorder of glycosylation that affects a restricted subset of glycoproteins. Transfection of MAGT1 mRNA enabled us to rescue proteins with defective glycosylation. Together, these data provide new clinical and pathophysiological foundations with important ramifications for the diagnosis and treatment of XMEN disease.

The role of nitric oxide (NO) in cancer progression has largely been studied in the context of tumor NOS2 expression. However, pro- versus anti-tumor signaling is also affected by tumor cell-macrophage interactions. While these cell-cell interactions are partly regulated by NO, the functional effects of NO flux on proinflammatory (M1) macrophages are unknown. Using a triple negative murine breast cancer model, we explored the potential role of macrophage Nos2 on 4T1 tumor progression. The effects of NO on macrophage phenotype were examined in bone marrow derived macrophages from wild type and Nos2 -/- mice following in vitro stimulation with cytokine/LPS combinations to produce low, medium, and high NO flux. Remarkably, Nos2 induction was spatially distinct, where Nos2 high cells expressed low cyclooxygenase-2 (Cox2) and vice versa. Importantly, in vitro M1 polarization with IFNγ+LPS induced high NO flux that was restricted to cells harboring depolarized mitochondria. This flux altered the magnitude and spatial extent of hypoxic gradients. Metabolic and single cell analyses demonstrated that single cell Nos2 induction limited the generation of hypoxic gradients in vitro, and Nos2-dependent and independent features may collaborate to regulate M1 functionality. It was found that Cox2 expression was important for Nos2 high cells to maintain NO tolerance. Furthermore, Nos2 and Cox2 expression in 4T1 mouse tumors was spatially orthogonal forming distinct cellular neighborhoods. In summary, the location and type of Nos2 high cells, NO flux, and the inflammatory status of other cells, such as Cox2 high cells in the tumor niche contribute to Nos2 inflammatory mechanisms that promote disease progression of 4T1 tumors., (Published by Elsevier B.V.)

Regulatory T (T reg) cells, a specialized subset of CD4 + T cells, are essential to prevent fatal autoimmunity. Expression of the T reg lineage-defining transcription factor Foxp3, and therefore their differentiation in the thymus, is dependent upon T cell receptor (TCR) and interleukin-2 (IL-2) signaling. Here, we report that the majority of IL-2-producing cells in the thymus are mature CD4 single-positive (CD4SP) thymocytes and that continuous IL-2 production sustained thymic T reg cell generation and control of systemic immune activation. Furthermore, single-cell RNA sequencing analysis of CD4 thymocyte subsets revealed that IL-2 was expressed in self-reactive CD4SP thymocytes, which also contain T reg precursor cells. Thus, our results suggest that the thymic T reg cell pool size is scaled by a key niche factor, IL-2, produced by self-reactive CD4SP thymocytes. This IL-2-dependent scaling of thymic T reg cell generation by overall self-reactivity of a mature post-selection thymic precursor pool may likely ensure adequate control of autoimmunity., (© 2019 Hemmers et al.)

We present a new method to directly quantify the dynamics of differentiation of multiple cellular subsets in unperturbed mice. We combine a pulse-chase protocol of 5-iodo-2'-deoxyuridine (IdU) injections with subsequent analysis by mass cytometry (CyTOF) and mathematical modeling of the IdU dynamics. Measurements by CyTOF allow for a wide range of cells to be analyzed at once, due to the availability of a large staining panel without the complication of fluorescence spillover. These are also compatible with direct detection of integrated iodine signal, with minimal impact on immunophenotyping based on the surface markers. Mathematical modeling beyond a binary classification of surface marker abundance allows for a continuum of cellular states as the cells transition from one state to another. Thus, we present a complete and robust method for directly quantifying differentiation at the systemic level, allowing for system-wide comparisons between different mouse strains and/or experimental conditions. Published 2019. This article is a U.S. Government work and is in the public domain in the USA., (Published 2019. This article is a U.S. Government work and is in the public domain in the USA.)

Type I interferons (IFN) are being rediscovered as potent anti-tumoral agents. Activation of the STimulator of INterferon Genes (STING) by DMXAA (5,6-dimethylxanthenone-4-acetic acid) can induce strong production of IFNα/β and rejection of transplanted primary tumors. In the present study, we address whether targeting STING with DMXAA also leads to the regression of spontaneous MMTV-PyMT mammary tumors. We show that these tumors are refractory to DMXAA-induced regression. This is due to a blockade in the phosphorylation of IRF3 and the ensuing IFNα/β production. Mechanistically, we identify TGFβ, which is abundant in spontaneous tumors, as a key molecule limiting this IFN-induced tumor regression by DMXAA. Finally, blocking TGFβ restores the production of IFNα by activated MHCII + tumor-associated macrophages, and enables tumor regression induced by STING activation. On the basis of these findings, we propose that type I IFN-dependent cancer therapies could be greatly improved by combinations including the blockade of TGFβ.

Near a bifurcation point, the response time of a system is expected to diverge due to the phenomenon of critical slowing down. We investigate critical slowing down in well-mixed stochastic models of biochemical feedback by exploiting a mapping to the mean-field Ising universality class. We analyze the responses to a sudden quench and to continuous driving in the model parameters. In the latter case, we demonstrate that our class of models exhibits the Kibble-Zurek collapse, which predicts the scaling of hysteresis in cellular responses to gradual perturbations. We discuss the implications of our results in terms of the tradeoff between a precise and a fast response. Finally, we use our mapping to quantify critical slowing down in T cells, where the addition of a drug is equivalent to a sudden quench in parameter space.

In cancer biology, the functional interpretation of genomic alterations is critical to achieve the promise of genomic profiling in the clinic. For chronic lymphocytic leukemia (CLL), a heterogeneous disease of B-lymphocytes maturing under constitutive B cell receptor (BCR) stimulation, the functional role of diverse clonal mutations remains largely unknown. Here, we demonstrate that alterations in BCR signaling dynamics underlie the progression of B cells toward malignancy. We reveal emergent dynamic features-bimodality, hypersensitivity, and hysteresis-in the BCR signaling pathway of primary CLL B cells. These signaling abnormalities in CLL quantitatively derive from BCR clustering and constitutive signaling with positive feedback reinforcement, as demonstrated through single-cell analysis of phospho-responses, computational modeling, and super-resolution imaging. Such dysregulated signaling segregates CLL patients by disease severity and clinical presentation. These findings provide a quantitative framework and methodology to assess complex and heterogeneous leukemia pathology and to inform therapeutic strategies in parallel with genomic profiling., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Thyroid cancer is the most common endocrine malignancy. Although an association between inflammation and thyroid cancer has long been recognized, a cause-effect relationship at the molecular level has yet to be elucidated. We explored how inflammation could contribute to thyroid carcinogenesis in Thrb PV/PV Pten +/- mice. The Thrb PV/PV Pten +/- mouse expresses a dominantly negative thyroid hormone receptor β (denoted as PV) and a deletion of one single allele of the Pten gene. This mutant mouse exhibits aggressive follicular thyroid cancer similarly as in patients. We found significantly increased infiltration of inflammatory monocytes in thyroid tumors of Thrb PV/PV Pten +/- mice, while no apparent changes in monocyte homeostasis in the bone marrow and blood of tumor-bearing mice. Using global gene expression profiling, we found altered expression of inflammation mediators in that the expression of Ptgs1, Sphk1, OPN, Chil1, Tnfrsf18, IL6, and Ccl12 genes was significantly increased and expression of Kit, Ly96, Ephx2, CD163, IL15, and Ccr2 was significantly decreased. Subsequent validation of the gene expression by mRNA analysis prompted us to further delineate the inflammatory role of osteopontin (OPN) in thyroid carcinogenesis because of its critical role in monocyte/macrophage functions and proinflammatory responses. We found that the protein abundance of OPN and its receptor, integrin β1, was highly increased and, concurrently, the downstream effectors AKT and NF-κB were significantly elevated to drive thyroid tumor progression of Thrb PV/PV Pten +/- mice. These results demonstrated that increased inflammation driven by elevated expression of immune-related genes and cytokines promoted thyroid cancer progression. Importantly, we uncovered OPN as a novel regulator in inflammatory response during thyroid carcinogenesis. These preclinical findings suggested that OPN can be a potential target for thyroid cancer therapy via modulation of inflammatory signaling., Competing Interests: None.

Intercellular communication mediated by cytokines is the main mechanism by which cells of the immune system talk to each other. Many aspects of cytokine signalling in the immune system have been explored in great detail at the structural, biophysical, biochemical and cellular levels. However, a systematic understanding of the quantitative rules that govern cytokine-mediated cell-to-cell communication is still lacking. Here, we discuss recent efforts in the field of systems immunology to bring about a quantitative understanding of cytokine-mediated communication between leukocytes and to provide novel insights into the orchestration of immune responses and inflammation.

We map a class of well-mixed stochastic models of biochemical feedback in steady state to the mean-field Ising model near the critical point. The mapping provides an effective temperature, magnetic field, order parameter, and heat capacity that can be extracted from biological data without fitting or knowledge of the underlying molecular details. We demonstrate this procedure on fluorescence data from mouse T cells, which reveals distinctions between how the cells respond to different drugs. We also show that the heat capacity allows inference of the absolute molecule number from fluorescence intensity. We explain this result in terms of the underlying fluctuations, and we demonstrate the generality of our work.

Recent efforts in systems immunology lead researchers to build quantitative models of cell activation and differentiation. One goal is to account for the distributions of proteins from single-cell measurements by flow cytometry or mass cytometry as readout of biological regulation. In that context, large cell-to-cell variability is often observed in biological quantities. We show here that these readouts, viewed in logarithmic scale may result in two easily-distinguishable modes, while the underlying distribution (in linear scale) is unimodal. We introduce a simple mathematical test to highlight this mismatch. We then dissect the flow of influence of cell-to-cell variability proposing a graphical model which motivates higher-dimensional analysis of the data. Finally we show how acquiring additional biological information can be used to reduce uncertainty introduced by cell-to-cell variability, helping to clarify whether the data is uni- or bimodal. This communication has cautionary implications for manual and automatic gating strategies, as well as clustering and modeling of single-cell measurements. © 2018 International Society for Advancement of Cytometry., (© 2018 International Society for Advancement of Cytometry.)

Concurrent amplifications of EGFR and PDGFRA have been reported in up to 5% of glioblastoma (GBM) and it remains unclear why such independent amplification events, and associated receptor overexpression, would be adaptive during glioma evolution. Here, we document that EGFR and PDGFRA protein co-expression occurs in 37% of GBM. There is wide cell-to-cell variation in the expressions of these receptor tyrosine kinases (RTKs) in stable tumor sphere lines, frequently defining tumor cell subpopulations with distinct sensitivities to growth factors and RTK inhibitors. We also find evidence for functional transactivation of PDGFRA by EGFR and EGF-induced receptor heterodimerization, both of which are abolished by EGFR inhibitors. These results indicate that GBM growth responses to targeted therapies previously tested in clinical trials are strongly influenced by the balance of EGFR and PDGFRA activation in individual cells, which is heterogeneous at baseline.

Immune cells constantly survey the host for pathogens or tumors and secrete cytokines to alert surrounding cells of these threats. In vivo, activated immune cells secrete cytokines for several hours, yet an acute immune reaction occurs over days. Given these divergent timescales, we addressed how cytokine-responsive cells translate brief cytokine exposure into phenotypic changes that persist over long timescales. We studied melanoma cell responses to transient exposure to the cytokine interferon γ (IFNγ) by combining a systems-scale analysis of gene expression dynamics with computational modeling and experiments. We discovered that IFNγ is captured by phosphatidylserine (PS) on the surface of viable cells both in vitro and in vivo then slowly released to drive long-term transcription of cytokine-response genes. This mechanism introduces an additional function for PS in dynamically regulating inflammation across diverse cancer and primary cell types and has potential to usher in new immunotherapies targeting PS and inflammatory pathways., (Published by Elsevier Inc.)

Immune cells communicate by exchanging cytokines to achieve a context-appropriate response, but the distances over which such communication happens are not known. Here, we used theoretical considerations and experimental models of immune responses in vitro and in vivo to quantify the spatial extent of cytokine communications in dense tissues. We established that competition between cytokine diffusion and consumption generated spatial niches of high cytokine concentrations with sharp boundaries. The size of these self-assembled niches scaled with the density of cytokine-consuming cells, a parameter that gets tuned during immune responses. In vivo, we measured interactions on length scales of 80-120 μm, which resulted in a high degree of cell-to-cell variance in cytokine exposure. Such heterogeneous distributions of cytokines were a source of non-genetic cell-to-cell variability that is often overlooked in single-cell studies. Our findings thus provide a basis for understanding variability in the patterning of immune responses by diffusible factors., (Published by Elsevier Inc.)

Despite progress in drug development, a quantitative and physiological understanding of how small-molecule inhibitors act on cells is lacking. Here, we measure the signalling and proliferative response of individual primary T-lymphocytes to a combination of antigen, cytokine and drug. We uncover two distinct modes of signalling inhibition: digital inhibition (the activated fraction of cells diminishes upon drug treatment, but active cells appear unperturbed), versus analogue inhibition (the activated fraction is unperturbed whereas activation response is diminished). We introduce a computational model of the signalling cascade that accounts for such inhibition dichotomy, and test the model predictions for the phenotypic variability of cellular responses. Finally, we demonstrate that the digital/analogue dichotomy of cellular response as revealed on short (signal transduction) timescales, translates into similar dichotomy on longer (proliferation) timescales. Our single-cell analysis of drug action illustrates the strength of quantitative approaches to translate in vitro pharmacology into functionally relevant cellular settings.

Lung adenocarcinomas with mutant epidermal growth factor receptor (EGFR) respond to EGFR-targeted tyrosine kinase inhibitors (TKIs), but resistance invariably occurs. We found that the Janus kinase (JAK)/signal transduction and activator of transcription 3 (STAT3) signaling pathway was aberrantly increased in TKI-resistant EGFR-mutant non-small cell lung cancer (NSCLC) cells. JAK2 inhibition restored sensitivity to the EGFR inhibitor erlotinib in TKI-resistant cell lines and xenograft models of EGFR-mutant TKI-resistant lung cancer. JAK2 inhibition uncoupled EGFR from its negative regulator, suppressor of cytokine signaling 5 (SOCS5), consequently increasing EGFR abundance and restoring the tumor cells' dependence on EGFR signaling. Furthermore, JAK2 inhibition led to heterodimerization of mutant and wild-type EGFR subunits, the activity of which was then blocked by TKIs. Our results reveal a mechanism whereby JAK2 inhibition overcomes acquired resistance to EGFR inhibitors and support the use of combination therapy with JAK and EGFR inhibitors for the treatment of EGFR-dependent NSCLC., (Copyright © 2016, American Association for the Advancement of Science.)

Current approaches to cancer immunotherapy aim to engage the natural T cell response against tumors. One limitation is the elimination of self-antigen-specific T cells from the immune repertoire. Using a system in which precursor frequency can be manipulated in a murine melanoma model, we demonstrated that the clonal abundance of CD4(+) T cells specific for self-tumor antigen positively correlated with antitumor efficacy. At elevated precursor frequencies, intraclonal competition impaired initial activation and overall expansion of the tumor-specific CD4(+) T cell population. However, through clonally derived help, this population acquired a polyfunctional effector phenotype and antitumor immunity was enhanced. Conversely, development of effector function was attenuated at low precursor frequencies due to irreversible T cell exhaustion. Our findings assert that the differential effects of T cell clonal abundance on phenotypic outcome should be considered during the design of adoptive T cell therapies, including use of engineered T cells., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Single-cell RNA and protein concentrations dynamically fluctuate because of stochastic ("noisy") regulation. Consequently, biological signaling and genetic networks not only translate stimuli with functional response but also random fluctuations. Intuitively, this feature manifests as the accumulation of fluctuations from the network source to the target. Taking advantage of the fact that noise propagates directionally, we developed a method for causation prediction that does not require time-lagged observations and therefore can be applied to data generated by destructive assays such as immunohistochemistry. Our method for causation prediction, "Inference of Network Directionality Using Covariance Elements (INDUCE)," exploits the theoretical relationship between a change in the strength of a causal interaction and the associated changes in the single cell measured entries of the covariance matrix of protein concentrations. We validated our method for causation prediction in two experimental systems where causation is well established: in an E. coli synthetic gene network, and in MEK to ERK signaling in mammalian cells. We report the first analysis of covariance elements documenting noise propagation from a kinase to a phosphorylated substrate in an endogenous mammalian signaling network.