Nuclear morphology is shaped by loop-extrusion programs.

It is well established that neutrophils adopt malleable polymorphonuclear shapes to migrate through narrow interstitial tissue spaces1-3. However, how polymorphonuclear structures are assembled remains unknown4. Here we show that in neutrophil progenitors, halting loop extrusion-a motor-powered process that generates DNA loops by pulling in chromatin5-leads to the assembly of polymorphonuclear genomes. Specifically, we found that in mononuclear neutrophil progenitors, acute depletion of the loop-extrusion loading factor nipped-B-like protein (NIPBL) induced the assembly of horseshoe, banded, ringed and hypersegmented nuclear structures and led to a reduction in nuclear volume, mirroring what is observed during the differentiation of neutrophils. Depletion of NIPBL also induced cell-cycle arrest, activated a neutrophil-specific gene program and conditioned a loss of interactions across topologically associating domains to generate a chromatin architecture that resembled that of differentiated neutrophils. Removing NIPBL resulted in enrichment for mega-loops and interchromosomal hubs that contain genes associated with neutrophil-specific enhancer repertoires and an inflammatory gene program. On the basis of these observations, we propose that in neutrophil progenitors, loop-extrusion programs produce lineage-specific chromatin architectures that permit the packing of chromosomes into geometrically confined lobular structures. Our data also provide a blueprint for the assembly of polymorphonuclear structures, and point to the possibility of engineering de novo nuclear shapes to facilitate the migration of effector cells in densely populated tumorigenic environments.

A system-wide health sciences faculty mentor training program is associated with improved effective mentoring and institutional climate.

Introduction: Mentorship is critical for faculty success, satisfaction, and engagement. However, many faculty, particularly underrepresented racial/ethnic (UR) faculty, lack access to high-quality mentoring. In an effort to improve mentoring for all faculty, we developed and implemented a formally structured faculty mentor training program (FMTP) across UC San Diego Health Sciences, which included institutional support, mentorship training, and department/division mentorship programs. Methods: FMTP impact was evaluated using three primary outcome variables: mentoring quality, mentoring behaviors, and institutional climate. Participants' self-assessed mentoring competencies were measured using validated instruments. Results: A total of 391 (23%) of Health Sciences faculty participated in FMTP. Participation rate was higher for women than men (30% versus 17%) and highest for UR faculty (39%). FMTP was implemented in 16 of 19 departments. Self-reported mentoring improved for FMTP participants with mentoring quality (p = 0.009) and meeting mentees' expectations (p = 0.01) continuing to improve for up to 2 years after training. However, participants were unsure if they were meeting UR mentees' expectations. FMTP participants were significantly more satisfied with mentoring quality (p < 0.001) compared to non-participants, with the greatest increase in satisfaction reported by UR faculty (38-61%). UR faculty reported improved overall morale (51-61%) and a perception that the environment was supportive for UR faculty (48-70%). Conclusion: The implementation of a system-wide formal structured FMTP was associated with improved faculty satisfaction, quality of mentoring, and institutional climate, especially for UR faculty.

Heterogeneity and clonal relationships of adaptive immune cells in ulcerative colitis revealed by single-cell analyses.

Inflammatory bowel disease (IBD) encompasses a spectrum of gastrointestinal disorders driven by dysregulated immune responses against gut microbiota. We integrated single-cell RNA and antigen receptor sequencing to elucidate key components, cellular states, and clonal relationships of the peripheral and gastrointestinal mucosal immune systems in health and ulcerative colitis (UC). UC was associated with an increase in IgG1+ plasma cells in colonic tissue, increased colonic regulatory T cells characterized by elevated expression of the transcription factor ZEB2, and an enrichment of a γδ T cell subset in the peripheral blood. Moreover, we observed heterogeneity in CD8+ tissue-resident memory T (TRM) cells in colonic tissue, with four transcriptionally distinct states of differentiation observed across health and disease. In the setting of UC, there was a marked shift of clonally related CD8+ TRM cells toward an inflammatory state, mediated, in part, by increased expression of the T-box transcription factor Eomesodermin. Together, these results provide a detailed atlas of transcriptional changes occurring in adaptive immune cells in the context of UC and suggest a role for CD8+ TRM cells in IBD.

Plasma Cell Fate Is Orchestrated by Elaborate Changes in Genome Compartmentalization and Inter-chromosomal Hubs.

The transition from the follicular B to the plasma cell stage is associated with large-scale changes in cell morphology. Here, we examine whether plasma cell development is also associated with changes in nuclear architecture. We find that the onset of plasma cell development is concomitant with a decline in remote genomic interactions; a gain in euchromatic character at loci encoding for factors that specify plasma cell fate, including Prdm1 and Atf4; and establishment of de novo inter-chromosomal hubs. We find that, in developing plasma cells and concurrent with transcriptional silencing, the Ebf1 locus repositions from an euchromatic to peri-centromeric heterochromatic environment. Finally, we find that inter-chromosomal hubs are enriched for the deposition of either H3K27Ac or H3K27me3. These data indicate that plasma cell fate is orchestrated by elaborate changes in genome topology and that epigenetic marks, linked with super-enhancers or transcriptionally repressed regions, are enriched at inter-chromosomal hubs.

mTORC1 coordinates an immediate unfolded protein response-related transcriptome in activated B cells preceding antibody secretion.

How activated B cells build biosynthetic pathways and organelle structures necessary for subsequent robust antibody secretion is still unclear. The dominant model holds that nascent plasma cells adapt to increased antibody synthesis by activating the unfolded protein response (UPR) under the control of the transcription factor Xbp1. Here, by analyzing gene expression in activated B cells with or without plasma cell-inductive signals, we find that follicular B cells up-regulate a wide array of UPR-affiliated genes before initiating antibody secretion; furthermore, initial transcription of these loci requires the mTORC1 kinase adaptor, Raptor, but not Xbp1. Transcriptomic analyses of resting marginal zone B cells, which generate plasma cells with exceptionally rapid kinetics, reinforce these results by revealing the basal expression of UPR-affiliated mRNA networks without detectable Xbp1 activity. We thus conclude that B cells utilize mTORC1 to prepare for subsequent plasma cell function, before the onset of antibody synthesis.

Molecular organization of mammalian meiotic chromosome axis revealed by expansion STORM microscopy.

During prophase I of meiosis, chromosomes become organized as loop arrays around the proteinaceous chromosome axis. As homologous chromosomes physically pair and recombine, the chromosome axis is integrated into the tripartite synaptonemal complex (SC) as this structure's lateral elements (LEs). While the components of the mammalian chromosome axis/LE-including meiosis-specific cohesin complexes, the axial element proteins SYCP3 and SYCP2, and the HORMA domain proteins HORMAD1 and HORMAD2-are known, the molecular organization of these components within the axis is poorly understood. Here, using expansion microscopy coupled with 2-color stochastic optical reconstruction microscopy (STORM) imaging (ExSTORM), we address these issues in mouse spermatocytes at a resolution of 10 to 20 nm. Our data show that SYCP3 and the SYCP2 C terminus, which are known to form filaments in vitro, form a compact core around which cohesin complexes, HORMADs, and the N terminus of SYCP2 are arrayed. Overall, our study provides a detailed structural view of the meiotic chromosome axis, a key organizational and regulatory component of meiotic chromosomes.

CHD4 is essential for transcriptional repression and lineage progression in B lymphopoiesis.

Cell lineage specification is a tightly regulated process that is dependent on appropriate expression of lineage and developmental stage-specific transcriptional programs. Here, we show that Chromodomain Helicase DNA-binding protein 4 (CHD4), a major ATPase/helicase subunit of Nucleosome Remodeling and Deacetylase Complexes (NuRD) in lymphocytes, is essential for specification of the early B cell lineage transcriptional program. In the absence of CHD4 in B cell progenitors in vivo, development of these cells is arrested at an early pro-B-like stage that is unresponsive to IL-7 receptor signaling and unable to efficiently complete V(D)J rearrangements at Igh loci. Our studies confirm that chromatin accessibility and transcription of thousands of gene loci are controlled dynamically by CHD4 during early B cell development. Strikingly, CHD4-deficient pro-B cells express transcripts of many non-B cell lineage genes, including genes that are characteristic of other hematopoietic lineages, neuronal cells, and the CNS, lung, pancreas, and other cell types. We conclude that CHD4 inhibits inappropriate transcription in pro-B cells. Together, our data demonstrate the importance of CHD4 in establishing and maintaining an appropriate transcriptome in early B lymphopoiesis via chromatin accessibility.

No strict requirement for eosinophils for bone marrow plasma cell survival.

Lasting antibody responses are maintained by long-lived plasma cells, which are thought to lodge in the BM in specialized survival niches. Eosinophils have been reported to function as a critical component of the BM survival niche where they are thought to provide pro-survival signals to nearby plasma cells. Recent study shows that many BM plasma cells are recently generated and chiefly short-lived cells, raising the possibility that rare plasma cell-eosinophil interactions are a rate-limiting step needed to establish lasting humoral immunity. To address these issues, we examined the impact of eosinophil depletion on short- and long-lived BM plasma cells in the context of antibody responses induced by both T-cell dependent and T-cell independent antigens. Surprisingly, our results failed to support a role for eosinophils in either plasma cell generation or survival. These studies included examination of plasma cell frequencies in mice lacking eosinophils either after antibody-mediated depletion, or due to mutation of the GATA1 locus.

mTOR has distinct functions in generating versus sustaining humoral immunity.

Little is known about the role of mTOR signaling in plasma cell differentiation and function. Furthermore, for reasons not understood, mTOR inhibition reverses antibody-associated disease in a murine model of systemic lupus erythematosus. Here, we have demonstrated that induced B lineage-specific deletion of the gene encoding RAPTOR, an essential signaling adaptor for rapamycin-sensitive mTOR complex 1 (mTORC1), abrogated the generation of antibody-secreting plasma cells in mice. Acute treatment with rapamycin recapitulated the effects of RAPTOR deficiency, and both strategies led to the ablation of newly formed plasma cells in the spleen and bone marrow while also obliterating preexisting germinal centers. Surprisingly, although perturbing mTOR activity caused a profound decline in serum antibodies that were specific for exogenous antigen or DNA, frequencies of long-lived bone marrow plasma cells were unaffected. Instead, mTORC1 inhibition led to decreased expression of immunoglobulin-binding protein (BiP) and other factors needed for robust protein synthesis. Consequently, blockade of antibody synthesis was rapidly reversed after termination of rapamycin treatment. We conclude that mTOR signaling plays critical but diverse roles in early and late phases of antibody responses and plasma cell differentiation.

Cellular and chromatin dynamics of antibody-secreting plasma cells.

Plasma cells are terminally differentiated B cells responsible for maintaining protective serum antibody titers. Despite their clinical importance, our understanding of the linear genomic features and chromatin structure of plasma cells is incomplete. The plasma cell differentiation program can be triggered by different signals and in multiple, diverse peripheral B cell subsets. This heterogeneity raises questions about the gene regulatory circuits required for plasma cell specification. Recently, new regulators of plasma cell differentiation have been identified and the enhancer landscapes of naïve B cells have been described. Other studies have revealed that the bone marrow niche harbors heterogeneous plasma cell subsets. Still undefined are the minimal requirements to become a plasma cell and what molecular features make peripheral B cell subsets competent to become antibody-secreting plasma cells. New technologies promise to reveal underlying chromatin configurations that promote efficient antibody secretion. For further resources related to this article, please visit the WIREs website.

AID targeting: old mysteries and new challenges.

Activation-induced cytidine deaminase (AID) mediates cytosine deamination and underlies two central processes in antibody diversification: somatic hypermutation and class-switch recombination. AID deamination is not exclusive to immunoglobulin loci; it can instigate DNA lesions in non-immunoglobulin genes and thus stringent checks are in place to constrain and restrict its activity. Recent findings have provided new insights into the mechanisms that target AID activity to specific genomic regions, revealing an involvement for noncoding RNAs associated with polymerase pausing and with enhancer transcription as well as genomic architecture. We review these findings and integrate them into a model for multilevel regulation of AID expression and targeting in immunoglobulin and non-immunoglobulin loci. Within this framework we discuss gaps in understanding, and outline important areas of further research.

Lasting antibody responses are mediated by a combination of newly formed and established bone marrow plasma cells drawn from clonally distinct precursors.

Current models hold that serum Ab titers are maintained chiefly by long-lived bone marrow (BM) plasma cells (PCs). In this study, we characterize the role of subpopulations of BM PCs in long-term humoral responses to T cell-dependent Ag. Surprisingly, our results indicate that 40-50% of BM PCs are recently formed cells, defined, in part, by rapid steady-state turnover kinetics and secretion of low-affinity IgM Abs. Further, for months after immunization with a hapten-protein conjugate, newly formed Ag-induced, IgM-secreting BM PCs were detected in parallel with longer-lived IgG-secreting cells, suggesting ongoing and parallel input to the BM PC pool from two distinct pools of activated B cells. Consistent with this interpretation, IgM and IgG Abs secreted by cells within distinct PC subsets exhibited distinct L chain usage. We conclude that long-term Ab responses are maintained by a dynamic BM PC pool composed of both recently formed and long-lived PCs drawn from clonally disparate precursors.

What is and what should always have been: long-lived plasma cells induced by T cell-independent antigens.

It is well accepted that Ag-induced B cell differentiation often results in the generation of exceptionally long-lived plasma cells. Much of the work supporting this viewpoint stems from studies focused on germinal center-derived plasma cells secreting high-affinity isotype-switched Abs in mice immunized with T cell-dependent Ags. In contrast, less attention has been devoted to understanding Ab responses to T cell-independent Ags and pathogens. In this study, we review recent work showing that T cell-independent Ags consisting of either polysaccharides or LPSs also induce the formation of long-lived plasma cells, despite their general inability to sustain germinal center responses. This new information provides a framework for more fully understanding the forces underlying immunity to pathogens that resist T cell recognition and the extracellular cues governing plasma cell longevity.

Long-lived bone marrow plasma cells are induced early in response to T cell-independent or T cell-dependent antigens.

The signals required to generate long-lived plasma cells remain unresolved. One widely cited model posits that long-lived plasma cells derive from germinal centers (GCs) in response to T cell-dependent (TD) Ags. Thus, T cell-independent (TI) Ags, which fail to sustain GCs, are considered ineffective at generating long-lived plasma cells. However, we show that long-lived hapten-specific plasma cells are readily induced without formation of GCs. Long-lived plasma cells developed in T cell-deficient mice after a single immunization with haptenated LPS, a widely used TI Ag. Long-lived plasma cells also formed in response to TD Ag when the GC response was experimentally prevented. These observations establish that long-lived plasma cells are induced in both TI and TD responses, and can arise independently of B cell maturation in GCs.