Loss of B1 and marginal zone B cells during ovarian cancer.

Recent advances in immunotherapy have not addressed the challenge presented by ovarian cancer. Although the peritoneum is an "accessible" locus for this disease there has been limited characterization of the immunobiology therein. We investigated the ID8-C57BL/6J ovarian cancer model and found marked depletion of B1 cells from the ascites of the peritoneal cavity. There was also selective loss of the B1 and marginal zone B cell subsets from the spleen. Immunity to antigens that activate these subsets validated their loss rather than relocation. A marked influx of myeloid-derived suppressor cells correlated with B cell subset depletion. These observations are discussed in the context of the housekeeping burden placed on innate B cells during ovarian cancer and to foster consideration of B cell biology in therapeutic strategies to address this challenge.

Intrinsically disordered domain of transcription factor TCF-1 is required for T cell developmental fidelity.

In development, pioneer transcription factors access silent chromatin to reveal lineage-specific gene programs. The structured DNA-binding domains of pioneer factors have been well characterized, but whether and how intrinsically disordered regions affect chromatin and control cell fate is unclear. Here, we report that deletion of an intrinsically disordered region of the pioneer factor TCF-1 (termed L1) leads to an early developmental block in T cells. The few T cells that develop from progenitors expressing TCF-1 lacking L1 exhibit lineage infidelity distinct from the lineage diversion of TCF-1-deficient cells. Mechanistically, L1 is required for activation of T cell genes and repression of GATA2-driven genes, normally reserved to the mast cell and dendritic cell lineages. Underlying this lineage diversion, L1 mediates binding of TCF-1 to its earliest target genes, which are subject to repression as T cells develop. These data suggest that the intrinsically disordered N terminus of TCF-1 maintains T cell lineage fidelity.

Quantitative control of Ets1 dosage by a multi-enhancer hub promotes Th1 cell differentiation and protects from allergic inflammation.

Multi-enhancer hubs are spatial clusters of enhancers present across numerous developmental programs. Here, we studied the functional relevance of these three-dimensional structures in T cell biology. Mathematical modeling identified a highly connected multi-enhancer hub at the Ets1 locus, comprising a noncoding regulatory element that was a hotspot for sequence variation associated with allergic disease in humans. Deletion of this regulatory element in mice revealed that the multi-enhancer connectivity was dispensable for T cell development but required for CD4+ T helper 1 (Th1) differentiation. These mice were protected from Th1-mediated colitis but exhibited overt allergic responses. Mechanistically, the multi-enhancer hub controlled the dosage of Ets1 that was required for CTCF recruitment and assembly of Th1-specific genome topology. Our findings establish a paradigm wherein multi-enhancer hubs control cellular competence to respond to an inductive cue through quantitative control of gene dosage and provide insight into how sequence variation within noncoding elements at the Ets1 locus predisposes individuals to allergic responses.

TCF-1 promotes chromatin interactions across topologically associating domains in T cell progenitors.

The high mobility group (HMG) transcription factor TCF-1 is essential for early T cell development. Although in vitro biochemical assays suggest that HMG proteins can serve as architectural elements in the assembly of higher-order nuclear organization, the contribution of TCF-1 on the control of three-dimensional (3D) genome structures during T cell development remains unknown. Here, we investigated the role of TCF-1 in 3D genome reconfiguration. Using gain- and loss-of-function experiments, we discovered that the co-occupancy of TCF-1 and the architectural protein CTCF altered the structure of topologically associating domains in T cell progenitors, leading to interactions between previously insulated regulatory elements and target genes at late stages of T cell development. The TCF-1-dependent gain in long-range interactions was linked to deposition of active enhancer mark H3K27ac and recruitment of the cohesin-loading factor NIPBL at active enhancers. These data indicate that TCF-1 has a role in controlling global genome organization during T cell development.

Single-cell multi-omics analysis of human pancreatic islets reveals novel cellular states in type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease in which immune cells destroy insulin-producing beta cells. The aetiology of this complex disease is dependent on the interplay of multiple heterogeneous cell types in the pancreatic environment. Here, we provide a single-cell atlas of pancreatic islets of 24 T1D, autoantibody-positive and nondiabetic organ donors across multiple quantitative modalities including ~80,000 cells using single-cell transcriptomics, ~7,000,000 cells using cytometry by time of flight and ~1,000,000 cells using in situ imaging mass cytometry. We develop an advanced integrative analytical strategy to assess pancreatic islets and identify canonical cell types. We show that a subset of exocrine ductal cells acquires a signature of tolerogenic dendritic cells in an apparent attempt at immune suppression in T1D donors. Our multimodal analyses delineate cell types and processes that may contribute to T1D immunopathogenesis and provide an integrative procedure for exploration and discovery of human pancreatic function.

IgD ligation allows peritoneal cavity B cell proliferation.

Atypical cytokine production and immune cell subset ratios, particularly those that include high proportions of macrophages, characterize tumor microenvironments (TMEs). TMEs can be modeled by culturing peritoneal cavity (PerC) cells which have a high macrophage to lymphocyte ratio. With TCR or BCR ligation, PerC lymphocyte proliferation is tempered by macrophages. However, PHA (T cells) and anti-CD40 (B cells) are activators that induce proliferation. Herein, we report that ligating IgD, in contrast to IgM, triggers PerC B cell proliferation. IL-4 addition enhanced the IgD response for BALB/c PerC B cells but suppressed that of C57BL/6 mice. Intriguingly, concurrent ligation of IgD and CD3ε rescued a PerC T cell proliferative response. These results serve to expand the list of targets for promoting cellular and humoral immunity in conditions that model macrophage-rich TMEs.

Transcription factors combine to paint the methylation landscape.

There is paucity of information about DNA methylation dynamics in immune cells. Roy et al. mapped the DNA methylation status of several thousand differentially methylated CpGs in human immune cells. They reported that the extent of cell type-specific hypermethylation is intriguingly most prevalent in adaptive immune cells rather than innate cells.

Foxp3 Re-distributes Its Heavy Lifting.

Understanding the mechanisms that establish regulatory T (Treg) cell identity is central to understanding Treg cell function. van der Veeken et al. now show that the lineage-determining transcription factor Foxp3 establishes Treg-cell-specific chromatin architecture indirectly, mostly by decreasing the expression of other transcriptional regulators, including TCF1.

Joint profiling of chromatin accessibility and CAR-T integration site analysis at population and single-cell levels.

Chimeric antigen receptor (CAR)-T immunotherapy has yielded impressive results in several B cell malignancies, establishing itself as a powerful means to redirect the natural properties of T lymphocytes. In this strategy, the T cell genome is modified by the integration of lentiviral vectors encoding CAR that direct tumor cell killing. However, this therapeutic approach is often limited by the extent of CAR-T cell expansion in vivo. A major outstanding question is whether or not CAR-T integration itself enhances the proliferative competence of individual T cells by rewiring their regulatory landscape. To address this question, it is critical to define the identity of an individual CAR-T cell and simultaneously chart where the CAR-T vector integrates into the genome. Here, we report the development of a method called EpiVIA (https://github.com/VahediLab/epiVIA) for the joint profiling of the chromatin accessibility and lentiviral integration site analysis at the population and single-cell levels. We validate our technique in clonal cells with previously defined integration sites and further demonstrate the ability to measure lentiviral integration sites and chromatin accessibility of host and viral genomes at the single-cell resolution in CAR-T cells. We anticipate that EpiVIA will enable the single-cell deconstruction of gene regulation during CAR-T therapy, leading to the discovery of cellular factors associated with durable treatment.

Genetic Variation in Type 1 Diabetes Reconfigures the 3D Chromatin Organization of T Cells and Alters Gene Expression.

Genetics is a major determinant of susceptibility to autoimmune disorders. Here, we examined whether genome organization provides resilience or susceptibility to sequence variations, and how this would contribute to the molecular etiology of an autoimmune disease. We generated high-resolution maps of linear and 3D genome organization in thymocytes of NOD mice, a model of type 1 diabetes (T1D), and the diabetes-resistant C57BL/6 mice. Multi-enhancer interactions formed at genomic regions harboring genes with prominent roles in T cell development in both strains. However, diabetes risk-conferring loci coalesced enhancers and promoters in NOD, but not C57BL/6 thymocytes. 3D genome mapping of NODxC57BL/6 F1 thymocytes revealed that genomic misfolding in NOD mice is mediated in cis. Moreover, immune cells infiltrating the pancreas of humans with T1D exhibited increased expression of genes located on misfolded loci in mice. Thus, genetic variation leads to altered 3D chromatin architecture and associated changes in gene expression that may underlie autoimmune pathology.

A mass spectrometry-based assay using metabolic labeling to rapidly monitor chromatin accessibility of modified histone proteins.

Histone post-translational modifications (PTMs) contribute to chromatin accessibility due to their chemical properties and their ability to recruit enzymes responsible for DNA readout and chromatin remodeling. To date, more than 400 different histone PTMs and thousands of combinations of PTMs have been identified, the vast majority with still unknown biological function. Identification and quantification of histone PTMs has become routine in mass spectrometry (MS) but, since raising antibodies for each PTM in a study can be prohibitive, lots of potential is lost from MS datasets when uncharacterized PTMs are found to be significantly regulated. We developed an assay that uses metabolic labeling and MS to associate chromatin accessibility with histone PTMs and their combinations. The labeling is achieved by spiking in the cell media a 5x concentration of stable isotope labeled arginine and allow cells to grow for at least one cell cycle. We quantified the labeling incorporation of about 200 histone peptides with a proteomics workflow, and we confirmed that peptides carrying PTMs with extensively characterized roles in active transcription or gene silencing were in highly or poorly labeled forms, respectively. Data were further validated using next-generation sequencing to assess the transcription rate of chromatin regions modified with five selected PTMs. Furthermore, we quantified the labeling rate of peptides carrying co-existing PTMs, proving that this method is suitable for combinatorial PTMs. We focus on the abundant bivalent mark H3K27me3K36me2, showing that H3K27me3 dominantly represses histone swapping rate even in the presence of the more permissive PTM H3K36me2. Together, we envision this method will help to generate hypotheses regarding histone PTM functions and, potentially, elucidate the role of combinatorial histone codes.

Oncogenic Notch Promotes Long-Range Regulatory Interactions within Hyperconnected 3D Cliques.

Chromatin loops enable transcription-factor-bound distal enhancers to interact with their target promoters to regulate transcriptional programs. Although developmental transcription factors such as active forms of Notch can directly stimulate transcription by activating enhancers, the effect of their oncogenic subversion on the 3D organization of cancer genomes is largely undetermined. By mapping chromatin looping genome-wide in Notch-dependent triple-negative breast cancer and B cell lymphoma, we show that beyond the well-characterized role of Notch as an activator of distal enhancers, Notch regulates its direct target genes by instructing enhancer repositioning. Moreover, a large fraction of Notch-instructed regulatory loops form highly interacting enhancer and promoter spatial clusters termed "3D cliques." Loss- and gain-of-function experiments show that Notch preferentially targets hyperconnected 3D cliques that regulate the expression of crucial proto-oncogenes. Our observations suggest that oncogenic hijacking of developmental transcription factors can dysregulate transcription through widespread effects on the spatial organization of cancer genomes.

PHA eludes macrophage suppression to activate CD8+ T cells.

Tumors may include a high proportion of immune modulatory cells and molecules that restrain the anti-cancer response. Activation of T cells to eliminate cancer cells within the immune-suppressive tumor microenvironment remains a challenge. We have shown that C57BL/6 J peritoneal cell culture models features of macrophage-dense tumors as TCR ligation fails to activate T cells unless IFNγ is neutralized or iNOS is inhibited. We tested other forms of T cell activation and found phytohemagglutinin (PHA) distinctive in the ability to markedly expand CD8 T cells in this model. IFNγ or iNOS inhibition was not necessary for this response. PHA triggered less IFNγ production and inhibitory PD-L1 expression than TCR ligation. Macrophages and CD44hi T cells bound PHA. Spleen T cell responses to PHA were markedly enhanced by the addition of peritoneal cells revealing that macrophages enhance T cell expansion. That PHA increases CD8 T cell responses within macrophage-dense culture suggests this mitogen might enhance anti-tumor immunity.

A mass spectrometry-based assay using metabolic labeling to rapidly monitor chromatin accessibility of modified histone proteins

Histone post-translational modifications (PTMs) contribute to chromatin accessibility due to their chemical properties and their ability to recruit enzymes responsible for DNA readout and chromatin remodeling. To date, more than 400 different histone PTMs and thousands of combinations of PTMs have been identified, the vast majority with still unknown biological function. Identification and quantification of histone PTMs has become routine in mass spectrometry (MS) but, since raising antibodies for each PTM in a study can be prohibitive, lots of potential is lost from MS datasets when uncharacterized PTMs are found to be significantly regulated. We developed an assay that uses metabolic labeling and MS to associate chromatin accessibility with histone PTMs and their combinations. The labeling is achieved by spiking in the cell media a 5x concentration of stable isotope labeled arginine and allow cells to grow for at least one cell cycle. We quantified the labeling incorporation of about 200 histone peptides with a proteomics workflow, and we confirmed that peptides carrying PTMs with extensively characterized roles in active transcription or gene silencing were in highly or poorly labeled forms, respectively. Data were further validated using next-generation sequencing to assess the transcription rate of chromatin regions modified with five selected PTMs. Furthermore, we quantified the labeling rate of peptides carrying co-existing PTMs, proving that this method is suitable for combinatorial PTMs. We focus on the abundant bivalent mark H3K27me3K36me2, showing that H3K27me3 dominantly represses histone swapping rate even in the presence of the more permissive PTM H3K36me2. Together, we envision this method will help to generate hypotheses regarding histone PTM functions and, potentially, elucidate the role of combinatorial histone codes.

A mass spectrometry-based assay using metabolic labeling to rapidly monitor chromatin accessibility of modified histone proteins

Histone post-translational modifications (PTMs) contribute to chromatin accessibility due to their chemical properties and their ability to recruit enzymes responsible for DNA readout and chromatin remodeling. To date, more than 400 different histone PTMs and thousands of combinations of PTMs have been identified, the vast majority with still unknown biological function. Identification and quantification of histone PTMs has become routine in mass spectrometry (MS) but, since raising antibodies for each PTM in a study can be prohibitive, lots of potential is lost from MS datasets when uncharacterized PTMs are found to be significantly regulated. We developed an assay that uses metabolic labeling and MS to associate chromatin accessibility with histone PTMs and their combinations. The labeling is achieved by spiking in the cell media a 5x concentration of stable isotope labeled arginine and allow cells to grow for at least one cell cycle. We quantified the labeling incorporation of about 200 histone peptides with a proteomics workflow, and we confirmed that peptides carrying PTMs with extensively characterized roles in active transcription or gene silencing were in highly or poorly labeled forms, respectively. Data were further validated using next-generation sequencing to assess the transcription rate of chromatin regions modified with five selected PTMs. Furthermore, we quantified the labeling rate of peptides carrying co-existing PTMs, proving that this method is suitable for combinatorial PTMs. We focus on the abundant bivalent mark H3K27me3K36me2, showing that H3K27me3 dominantly represses histone swapping rate even in the presence of the more permissive PTM H3K36me2. Together, we envision this method will help to generate hypotheses regarding histone PTM functions and, potentially, elucidate the role of combinatorial histone codes.

High macrophage PD-L1 expression not responsible for T cell suppression.

Tumors are often comprised of microenvironments (TMEs) with a high proportion of cells and molecules that regulate immunity. Peritoneal cavity (PerC) cell culture reproduces key features of TMEs as lymphocyte proliferation is suppressed by PerC macrophages (Mϕs). We monitored the expression of T cell stimulatory (Class II MHC, B7) and inhibitory (PD-L1) molecules by PerC APCs before and after culture and report here that IFNγ-driven PD-L1 expression increased markedly on PerC Mϕs after TCR ligation, even more so than seen with direct APC activation by LPS. Considering the high APC composition of and pronounced PD-L1 expression by PerC cells, it was surprising that blocking PD-1/PD-L1 interaction by mAb neutralization or genetic ablation did not relieve suppression. This result parallels TME challenges observed in the clinic and validates the need for further study of this culture model to inform strategies to promote anti-tumor immunity.

Macrophage regulation of B cell proliferation.

Unlike organized lymphoid tissue, the tumor microenvironment (TME) often includes a high proportion of immunosuppressive macrophages. We model the TME by culturing peritoneal cavity (PerC) cells that naturally have a high macrophage to lymphocyte ratio. Prior studies revealed that, following TCR ligation, PerC T cell proliferation is suppressed due to IFNγ-triggered inducible nitric oxide synthase expression. In this study we assessed the ability of PerC B cells to respond to surrogate activating signals in the presence of high numbers of macrophages. Surface IgM (BCR) ligation led to cyclooxygenase-mediated, and TLR-4 ligation to IL10-mediated, suppression of PerC B cell proliferation. In contrast, PerC B cells had a robust response to CD40 ligation, which could overcome the suppression generated by the BCR or TLR-4 response. However, the CD40 response was suppressed by concurrent TCR ligation. These results reveal the challenges of promoting B and T cell responses in macrophage-rich conditions that model the TME.

Erythropoietin increases macrophage-mediated T cell suppression.

Erythropoietin (EPO), used to treat anemia in cancer patients, has been reported to accelerate tumor progression and increase mortality. Research of the mechanism for this effect has focused upon EPOR expression by tumor cells. We model the high macrophage to lymphocyte ratio found in tumor microenvironments (TMEs) by culturing peritoneal cavity (PerC) cells that naturally have a high macrophage to T cell ratio. Following TCR ligation, C57BL/6J PerC T cell proliferation is suppressed due to IFNγ-triggered inducible nitric oxide synthase (iNOS) expression. EPO was tested in the PerC culture model and found to increase T cell suppression. This effect could be abrogated by inhibiting iNOS by enzyme inhibition, genetic ablation, or blocking IFNγ signaling. Flow cytometry revealed the EPOR on CD11b(+)F4/80(+) macrophages. These results suggest that EPO could increase T cell suppression in the TME by acting directly on macrophages.