A Perfect Storm Averted: Flawed Systems, a Dropped Ball, and Cognitive Biases Delay a Critical Diagnosis.

This is the first case of Cancer Morbidity, Mortality, and Improvement Rounds, a series of articles intended to explore the unique safety risks experienced by oncology patients through the lens of quality improvement, systems and human factors engineering, and cognitive psychology. This case highlights how multiple overlapping factors contributed to a delay in diagnosing disseminated tuberculosis in a patient with lung cancer. The discussion focuses on the ways that cognitive biases contributed to the delayed diagnosis in a patient who, with the benefit of hindsight, exhibited several signs and symptoms suggesting tuberculosis.Cancer Morbidity, Mortality, and Improvement Rounds is a series of articles intended to explore the unique safety risks experienced by oncology patients through the lens of quality improvement, systems and human factors engineering, and cognitive psychology. For purposes of clarity, each case focuses on a single theme, although, as is true for all medical incidents, there are almost always multiple, overlapping, contributing factors. The quality improvement paradigm used here, which focuses on root cause analyses and opportunities to improve care delivery systems, was previously outlined in this journal.1.

Cancer vaccines: Building a bridge over troubled waters.

Cancer vaccines aim to direct the immune system to eradicate cancer cells. Here we review the essential immunologic concepts underpinning natural immunity and highlight the multiple unique challenges faced by vaccines targeting cancer. Recent technological advances in mass spectrometry, neoantigen prediction, genetically and pharmacologically engineered mouse models, and single-cell omics have revealed new biology, which can help to bridge this divide. We particularly focus on translationally relevant aspects, such as antigen selection and delivery and the monitoring of human post-vaccination responses, and encourage more aggressive exploration of novel approaches.

Improved T-cell Immunity Following Neoadjuvant Chemotherapy in Ovarian Cancer.

PURPOSE: Although local tissue-based immune responses are critical for elucidating direct tumor-immune cell interactions, peripheral immune responses are increasingly recognized as occupying an important role in anticancer immunity. We evaluated serial blood samples from patients with advanced epithelial ovarian cancer (EOC) undergoing standard-of-care neoadjuvant carboplatin and paclitaxel chemotherapy (including dexamethasone for prophylaxis of paclitaxel-associated hypersensitivity reactions) to characterize the evolution of the peripheral immune cell function and composition across the course of therapy. EXPERIMENTAL DESIGN: Serial blood samples from 10 patients with advanced high-grade serous ovarian cancer treated with neoadjuvant chemotherapy (NACT) were collected before the initiation of chemotherapy, after the third and sixth cycles, and approximately 2 months after completion of chemotherapy. T-cell function was evaluated using ex vivo IFNγ ELISpot assays, and the dynamics of T-cell repertoire and immune cell composition were assessed using bulk and single-cell RNA sequencing (RNAseq). RESULTS: T cells exhibited an improved response to viral antigens after NACT, which paralleled the decrease in CA125 levels. Single-cell analysis revealed increased numbers of memory T-cell receptor (TCR) clonotypes and increased central memory CD8+ and regulatory T cells throughout chemotherapy. Finally, administration of NACT was associated with increased monocyte frequency and expression of HLA class II and antigen presentation genes; single-cell RNAseq analyses showed that although driven largely by classical monocytes, increased class II gene expression was a feature observed across monocyte subpopulations after chemotherapy. CONCLUSIONS: NACT may alleviate tumor-associated immunosuppression by reducing tumor burden and may enhance antigen processing and presentation. These findings have implications for the successful combinatorial applications of immune checkpoint blockade and therapeutic vaccine approaches in EOC.

Ikaros Is a Negative Regulator of B1 Cell Development and Function.

B1 B cells secrete most of the circulating natural antibodies and are considered key effector cells of the innate immune response. However, B1 cell-associated antibodies often cross-react with self-antigens, which leads to autoimmunity, and B1 cells have been implicated in cancer. How B1 cell activity is regulated remains unclear. We show that the Ikaros transcription factor is a major negative regulator of B1 cell development and function. Using conditional knock-out mouse models to delete Ikaros at different locations, we show that Ikaros-deficient mice exhibit specific and significant increases in splenic and bone marrow B1 cell numbers, and that the B1 progenitor cell pool is increased ∼10-fold in the bone marrow. Ikaros-null B1 cells resemble WT B1 cells at the molecular and cellular levels, but show a down-regulation of signaling components important for inhibiting proliferation and immunoglobulin production. Ikaros-null B1 cells hyper-react to TLR4 stimulation and secrete high amounts of IgM autoantibodies. These results indicate that Ikaros is required to limit B1 cell homeostasis in the adult.

Ikaros limits follicular B cell activation by regulating B cell receptor signaling pathways.

The Ikaros transcription factor is essential for early B cell development, but its effect on mature B cells is debated. We show that Ikaros is required to limit the response of naive splenic B cells to B cell receptor signals. Ikaros deficient follicular B cells grow larger and enter cell cycle faster after anti-IgM stimulation. Unstimulated mutant B cells show deregulation of positive and negative regulators of signal transduction at the mRNA level, and constitutive phosphorylation of ERK, p38, SYK, BTK, AKT and LYN. Stimulation results in enhanced and prolonged ERK and p38 phosphorylation, followed by hyper-proliferation. Pharmacological inhibition of ERK and p38 abrogates the increased proliferative response of Ikaros deficient cells. These results suggest that Ikaros functions as a negative regulator of follicular B cell activation.

Regulation of DNA methylation dictates Cd4 expression during the development of helper and cytotoxic T cell lineages.

During development, progenitor cells with binary potential give rise to daughter cells that have distinct functions. Heritable epigenetic mechanisms then lock in gene-expression programs that define lineage identity. Regulation of the gene encoding the T cell-specific coreceptor CD4 in helper and cytotoxic T cells exemplifies this process, with enhancer- and silencer-regulated establishment of epigenetic memory for stable gene expression and repression, respectively. Using a genetic screen, we identified the DNA-methylation machinery as essential for maintaining silencing of Cd4 in the cytotoxic lineage. Furthermore, we found a requirement for the proximal enhancer in mediating the removal of DNA-methylation marks from Cd4, which allowed stable expression of Cd4 in helper T cells. Our findings suggest that stage-specific methylation and demethylation events in Cd4 regulate its heritable expression in response to the distinct signals that dictate lineage 'choice' during T cell development.

A validated regulatory network for Th17 cell specification.

Th17 cells have critical roles in mucosal defense and are major contributors to inflammatory disease. Their differentiation requires the nuclear hormone receptor RORγt working with multiple other essential transcription factors (TFs). We have used an iterative systems approach, combining genome-wide TF occupancy, expression profiling of TF mutants, and expression time series to delineate the Th17 global transcriptional regulatory network. We find that cooperatively bound BATF and IRF4 contribute to initial chromatin accessibility and, with STAT3, initiate a transcriptional program that is then globally tuned by the lineage-specifying TF RORγt, which plays a focal deterministic role at key loci. Integration of multiple data sets allowed inference of an accurate predictive model that we computationally and experimentally validated, identifying multiple new Th17 regulators, including Fosl2, a key determinant of cellular plasticity. This interconnected network can be used to investigate new therapeutic approaches to manipulate Th17 functions in the setting of inflammatory disease.

Dynamic microRNA gene transcription and processing during T cell development.

By disrupting microRNA (miRNA) biogenesis, we previously showed that this pathway is critical for the differentiation and function of T cells. Although various cloning studies have shown that many miRNAs are expressed during T cell development, and in a dynamic manner, it was unclear how comprehensive these earlier analyses were. We therefore decided to profile miRNA expression by next generation sequencing. Furthermore, we profiled miRNA expression starting from the hematopoietic stem cell. This analysis revealed that miRNA expression during T cell development is extremely dynamic, with 645 miRNAs sequenced, and the expression of some varying by as much as 3 orders of magnitude. Furthermore, changes in precursor processing led to altered mature miRNA sequences. We also analyzed the structures of the primary miRNA transcripts expressed in T cells and found that many were extremely long. The longest was pri-mir-29b-1/29a at ∼168 kb. All the long pri-miRNAs also displayed extensive splicing. Our findings indicate that miRNA expression during T cell development is both a highly dynamic and a highly regulated process.

The epigenetic landscape of lineage choice: lessons from the heritability of CD4 and CD8 expression.

Developing αß T cells choose between the helper and cytotoxic lineages, depending upon the specificity of their T cell receptors for MHC molecules. The expression of the CD4 co-receptor on helper cells and the CD8 co-receptor on cytotoxic cells is intimately linked to this decision, and their regulation at the transcriptional level has been the subject of intense study to better understand lineage choice. Indeed, as the fate of developing T cells is decided, the expression status of these genes is accordingly locked. Genetic models have revealed important transcriptional elements and the ability to manipulate these elements in the framework of development has added a new perspective on the temporal nature of their function and the epigenetic maintenance of gene expression. We examine here novel insights into epigenetic mechanisms that have arisen through the study of these genes.

Ikaros in B cell development and function.

The zinc finger transcription factor, Ikaros, is a central regulator of hematopoiesis. It is required for the development of the earliest B cell progenitors and at later stages for VDJ recombination and B cell receptor expression. Mature B cells rely on Ikaros to set the activation threshold for various stimuli, and to choose the correct antibody isotype during class switch recombination. Thus, Ikaros contributes to nearly every level of B cell differentiation and function.

RUNX transcription factor-mediated association of Cd4 and Cd8 enables coordinate gene regulation.

T cell fate is associated with mutually exclusive expression of CD4 or CD8 in helper and cytotoxic T cells, respectively. How expression of one locus is temporally coordinated with repression of the other has been a long-standing enigma, though we know RUNX transcription factors activate the Cd8 locus, silence the Cd4 locus, and repress the Zbtb7b locus (encoding the transcription factor ThPOK), which is required for CD4 expression. Here we found that nuclear organization was altered by interplay among members of this transcription factor circuitry: RUNX binding mediated association of Cd4 and Cd8 whereas ThPOK binding kept the loci apart. Moreover, targeted deletions within Cd4 modulated CD8 expression and pericentromeric repositioning of Cd8. Communication between Cd4 and Cd8 thus appears to enable long-range epigenetic regulation to ensure that expression of one excludes the other in mature CD4 or CD8 single-positive (SP) cells.

Ikaros controls isotype selection during immunoglobulin class switch recombination.

Class switch recombination (CSR) allows the humoral immune response to exploit different effector pathways through specific secondary antibody isotypes. However, the molecular mechanisms and factors that control immunoglobulin (Ig) isotype choice for CSR are unclear. We report that deficiency for the Ikaros transcription factor results in increased and ectopic CSR to IgG(2b) and IgG(2a), and reduced CSR to all other isotypes, regardless of stimulation. Ikaros suppresses active chromatin marks, transcription, and activation-induced cytidine deaminase (AID) accessibility at the gamma2b and gamma2a genes to inhibit class switching to these isotypes. Further, Ikaros directly regulates isotype gene transcription as it directly binds the Igh 3' enhancer and interacts with isotype gene promoters. Finally, Ikaros-mediated repression of gamma2b and gamma2a transcription promotes switching to other isotype genes by allowing them to compete for AID-mediated recombination at the single-cell level. Thus, our results reveal transcriptional competition between constant region genes in individual cells to be a critical and general mechanism for isotype specification during CSR. We show that Ikaros is a master regulator of this competition.

Ikaros represses the transcriptional response to Notch signaling in T-cell development.

Notch activity is essential for early T-cell differentiation, but aberrant activity induces T-cell transformation. Thus, Notch target genes must be efficiently silenced in cells where Notch activity is no longer required. How these genes are repressed remains poorly understood. We report here that the Ikaros transcription factor plays a crucial role in repressing the transcriptional response to Notch signaling in T-cell development. Using the Notch target gene Hes-1 as a model, we show that Ikaros and RBP-Jkappa, the transcriptional mediator of Notch signaling, compete for binding to two elements in the Hes-1 promoter in immature thymocytes. This antagonistic interaction likely occurs at the CD4(-) CD8(-) CD3(-) double-negative 4 (DN4) stage, where Ikaros levels and binding to the Hes-1 promoter increase sharply and wild-type thymocytes lose their capacity to transcribe Hes-1 upon Notch stimulation. Nonresponsiveness to Notch signaling requires Ikaros, as Ikaros-deficient DN4 and CD4(+) CD8(+) double-positive (DP) cells remain competent to express Hes-1 after Notch activation. Further, Hes-1 promoter sequences from Ikaros-deficient DP cells show reduced trimethylated H3K27, a modification associated with silent chromatin. These results indicate that Ikaros functions as a transcriptional checkpoint to repress Notch target gene expression in T cells.

Novel insights into the relationships between dendritic cell subsets in human and mouse revealed by genome-wide expression profiling.

BACKGROUND: Dendritic cells (DCs) are a complex group of cells that play a critical role in vertebrate immunity. Lymph-node resident DCs (LN-DCs) are subdivided into conventional DC (cDC) subsets (CD11b and CD8alpha in mouse; BDCA1 and BDCA3 in human) and plasmacytoid DCs (pDCs). It is currently unclear if these various DC populations belong to a unique hematopoietic lineage and if the subsets identified in the mouse and human systems are evolutionary homologs. To gain novel insights into these questions, we sought conserved genetic signatures for LN-DCs and in vitro derived granulocyte-macrophage colony stimulating factor (GM-CSF) DCs through the analysis of a compendium of genome-wide expression profiles of mouse or human leukocytes. RESULTS: We show through clustering analysis that all LN-DC subsets form a distinct branch within the leukocyte family tree, and reveal a transcriptomal signature evolutionarily conserved in all LN-DC subsets. Moreover, we identify a large gene expression program shared between mouse and human pDCs, and smaller conserved profiles shared between mouse and human LN-cDC subsets. Importantly, most of these genes have not been previously associated with DC function and many have unknown functions. Finally, we use compendium analysis to re-evaluate the classification of interferon-producing killer DCs, lin-CD16+HLA-DR+ cells and in vitro derived GM-CSF DCs, and show that these cells are more closely linked to natural killer and myeloid cells, respectively. CONCLUSION: Our study provides a unique database resource for future investigation of the evolutionarily conserved molecular pathways governing the ontogeny and functions of leukocyte subsets, especially DCs.

Immature CD4+CD8+ thymocytes and mature T cells regulate Nur77 distinctly in response to TCR stimulation.

The orphan steroid receptor, Nur77, is thought to be a central participant in events leading to TCR-mediated clonal deletion of immature thymocytes. Interestingly, although both immature and mature murine T cell populations rapidly up-regulate Nur77 after TCR stimulation, immature CD4+CD8+ thymocytes respond by undergoing apoptosis, whereas their mature descendants respond by dividing. To understand these developmental differences in susceptibility to the proapoptotic potential of Nur77, we compared its regulation and compartmentalization and show that mature, but not immature, T cells hyperphosphorylate Nur77 in response to TCR signals. Nur77 resides in the nucleus of immature CD4+CD8+ thymocytes throughout the course of its expression and is not found in either the organellar or cytoplasmic fractions. However, hyperphosphorylation of Nur77 in mature T cells, which is mediated by both the MAPK and PI3K/Akt pathways, shifts its localization from the nucleus to the cytoplasm. The failure of immature CD4+CD8+ thymocytes to hyperphosphorylate Nur77 in response to TCR stimulation may be due in part to decreased Akt activity at this developmental stage.

Notch activation is an early and critical event during T-Cell leukemogenesis in Ikaros-deficient mice.

The Ikaros transcription factor is both a key regulator of lymphocyte differentiation and a tumor suppressor in T lymphocytes. Mice carrying a hypomorphic mutation (Ik(L/L)) in the Ikaros gene all develop thymic lymphomas. Ik(L/L) tumors always exhibit strong activation of the Notch pathway, which is required for tumor cell proliferation in vitro. Notch activation occurs early in tumorigenesis and may precede transformation, as ectopic expression of the Notch targets Hes-1 and Deltex-1 is detected in thymocytes from young Ik(L/L) mice with no overt signs of transformation. Notch activation is further amplified by secondary mutations that lead to C-terminal truncations of Notch 1. Strikingly, restoration of Ikaros activity in tumor cells leads to a rapid and specific downregulation of Notch target gene expression and proliferation arrest. Furthermore, Ikaros binds to the Notch-responsive element in the Hes-1 promoter and represses Notch-dependent transcription from this promoter. Thus, Ikaros-mediated repression of Notch target gene expression may play a critical role in defining the tumor suppressor function of this factor.

Cutting edge: identification of the targets of clonal deletion in an unmanipulated thymus.

Autoreactive thymocytes can be eliminated by clonal deletion during their development in the thymus. The precise developmental stage(s) at which clonal deletion occurs in a normal thymus has been difficult to assess, in large part because of the absence of a specific marker for TCR-mediated apoptosis. In this report, we reveal that Nur77 expression can be used as a specific marker of clonal deletion in an unmanipulated thymus and directly identify TCRintCD4+CD8+ and semimature CD4+CD8- thymocytes as the principal targets of deletion. These data indicate that clonal deletion normally occurs at a relatively late stage of development, as cells mature from CD4+CD8+ thymocytes to single-positive T cells.