Lineage-specific 3D genome organization is assembled at multiple scales by IKAROS.

A generic level of chromatin organization generated by the interplay between cohesin and CTCF suffices to limit promiscuous interactions between regulatory elements, but a lineage-specific chromatin assembly that supersedes these constraints is required to configure the genome to guide gene expression changes that drive faithful lineage progression. Loss-of-function approaches in B cell precursors show that IKAROS assembles interactions across megabase distances in preparation for lymphoid development. Interactions emanating from IKAROS-bound enhancers override CTCF-imposed boundaries to assemble lineage-specific regulatory units built on a backbone of smaller invariant topological domains. Gain of function in epithelial cells confirms IKAROS' ability to reconfigure chromatin architecture at multiple scales. Although the compaction of the Igκ locus required for genome editing represents a function of IKAROS unique to lymphocytes, the more general function to preconfigure the genome to support lineage-specific gene expression and suppress activation of extra-lineage genes provides a paradigm for lineage restriction.

BRWD1 orchestrates small pre-B cell chromatin topology by converting static to dynamic cohesin.

Lymphocyte development consists of sequential and mutually exclusive cell states of proliferative selection and antigen receptor gene recombination. Transitions between each state require large, coordinated changes in epigenetic landscapes and transcriptional programs. How this occurs remains unclear. Here we demonstrate that in small pre-B cells, the lineage and stage-specific epigenetic reader bromodomain and WD repeat-containing protein 1 (BRWD1) reorders three-dimensional chromatin topology to affect the transition between proliferative and gene recombination molecular programs. BRWD1 regulated the switch between poised and active enhancers interacting with promoters, and coordinated this switch with Igk locus contraction. BRWD1 did so by converting chromatin-bound static to dynamic cohesin competent to mediate long-range looping. ATP-depletion revealed cohesin conversion to be the main energetic mechanism dictating dynamic chromatin looping. Our findings provide a new mechanism of cohesin regulation and reveal how cohesin function can be dictated by lineage contextual mechanisms to facilitate specific cell fate transitions.

The endogenous repertoire harbors self-reactive CD4+ T cell clones that adopt a follicular helper T cell-like phenotype at steady state.

The T cell repertoire of healthy mice and humans harbors self-reactive CD4+ conventional T (Tconv) cells capable of inducing autoimmunity. Using T cell receptor profiling paired with in vivo clonal analysis of T cell differentiation, we identified Tconv cell clones that are recurrently enriched in non-lymphoid organs following ablation of Foxp3+ regulatory T (Treg) cells. A subset of these clones was highly proliferative in the lymphoid organs at steady state and exhibited overt reactivity to self-ligands displayed by dendritic cells, yet were not purged by clonal deletion. These clones spontaneously adopted numerous hallmarks of follicular helper T (TFH) cells, including expression of Bcl6 and PD-1, exhibited an elevated propensity to localize within B cell follicles at steady state, and produced interferon-γ in non-lymphoid organs following sustained Treg cell depletion. Our work identifies a naturally occurring population of self-reactive TFH-like cells and delineates a previously unappreciated fate for self-specific Tconv cells.

Novel specialized cell state and spatial compartments within the germinal center.

Within germinal centers (GCs), complex and highly orchestrated molecular programs must balance proliferation, somatic hypermutation and selection to both provide effective humoral immunity and to protect against genomic instability and neoplastic transformation. In contrast to this complexity, GC B cells are canonically divided into two principal populations, dark zone (DZ) and light zone (LZ) cells. We now demonstrate that, following selection in the LZ, B cells migrated to specialized sites within the canonical DZ that contained tingible body macrophages and were sites of ongoing cell division. Proliferating DZ (DZp) cells then transited into the larger DZ to become differentiating DZ (DZd) cells before re-entering the LZ. Multidimensional analysis revealed distinct molecular programs in each population commensurate with observed compartmentalization of noncompatible functions. These data provide a new three-cell population model that both orders critical GC functions and reveals essential molecular programs of humoral adaptive immunity.

CXCR4 signaling directs Igk recombination and the molecular mechanisms of late B lymphopoiesis.

In B lymphopoiesis, activation of the pre-B cell antigen receptor (pre-BCR) is associated with both cell cycle exit and Igk recombination. Yet how the pre-BCR mediates these functions remains unclear. Here, we demonstrate that the pre-BCR initiates a feed-forward amplification loop mediated by the transcription factor interferon regulatory factor 4 and the chemokine receptor C-X-C motif chemokine receptor 4 (CXCR4). CXCR4 ligation by C-X-C motif chemokine ligand 12 activates the mitogen-activated protein kinase extracellular-signal-regulated kinase, which then directs the development of small pre- and immature B cells, including orchestrating cell cycle exit, pre-BCR repression, Igk recombination and BCR expression. In contrast, pre-BCR expression and escape from interleukin-7 have only modest effects on B cell developmental transcriptional and epigenetic programs. These data show a direct and central role for CXCR4 in orchestrating late B cell lymphopoiesis. Furthermore, in the context of previous findings, our data provide a three-receptor system sufficient to recapitulate the essential features of B lymphopoiesis in vitro.

Quantifying in situ adaptive immune cell cognate interactions in humans.

Two-photon excitation microscopy (TPEM) has revolutionized the understanding of adaptive immunity. However, TPEM usually requires animal models and is not amenable to the study of human disease. The recognition of antigen by T cells requires cell contact and is associated with changes in T cell shape. We postulated that by capturing these features in fixed tissue samples, we could quantify in situ adaptive immunity. Therefore, we used a deep convolutional neural network to identify fundamental distance and cell-shape features associated with cognate help (cell-distance mapping (CDM)). In mice, CDM was comparable to TPEM in discriminating cognate T cell-dendritic cell (DC) interactions from non-cognate T cell-DC interactions. In human lupus nephritis, CDM confirmed that myeloid DCs present antigen to CD4+ T cells and identified plasmacytoid DCs as an important antigen-presenting cell. These data reveal a new approach with which to study human in situ adaptive immunity broadly applicable to autoimmunity, infection, and cancer.

RAG Represents a Widespread Threat to the Lymphocyte Genome.

The RAG1 endonuclease, together with its cofactor RAG2, is essential for V(D)J recombination but is a potent threat to genome stability. The sources of RAG1 mis-targeting and the mechanisms that have evolved to suppress it are poorly understood. Here, we report that RAG1 associates with chromatin at thousands of active promoters and enhancers in the genome of developing lymphocytes. The mouse and human genomes appear to have responded by reducing the abundance of "cryptic" recombination signals near RAG1 binding sites. This depletion operates specifically on the RSS heptamer, whereas nonamers are enriched at RAG1 binding sites. Reversing this RAG-driven depletion of cleavage sites by insertion of strong recombination signals creates an ectopic hub of RAG-mediated V(D)J recombination and chromosomal translocations. Our findings delineate rules governing RAG binding in the genome, identify areas at risk of RAG-mediated damage, and highlight the evolutionary struggle to accommodate programmed DNA damage in developing lymphocytes.

Self-reactive IgE exacerbates interferon responses associated with autoimmunity.

Canonically, immunoglobulin E (IgE) mediates allergic immune responses by triggering mast cells and basophils to release histamine and type 2 helper cytokines. Here we found that in human systemic lupus erythematosus (SLE), IgE antibodies specific for double-stranded DNA (dsDNA) activated plasmacytoid dendritic cells (pDCs), a type of cell of the immune system linked to viral defense, which led to the secretion of substantial amounts of interferon-α (IFN-α). The concentration of dsDNA-specific IgE found in patient serum correlated with disease severity and greatly potentiated pDC function by triggering phagocytosis via the high-affinity FcɛRI receptor for IgE, followed by Toll-like receptor 9 (TLR9)-mediated sensing of DNA in phagosomes. Our findings expand the known pathogenic mechanisms of IgE-mediated inflammation beyond those found in allergy and demonstrate that IgE can trigger interferon responses capable of exacerbating self-destructive autoimmune responses.

Histone reader BRWD1 targets and restricts recombination to the Igk locus.

B lymphopoiesis requires that immunoglobulin genes be accessible to RAG1-RAG2 recombinase. However, the RAG proteins bind widely to open chromatin, which suggests that additional mechanisms must restrict RAG-mediated DNA cleavage. Here we show that developmental downregulation of interleukin 7 (IL-7)-receptor signaling in small pre-B cells induced expression of the bromodomain-family member BRWD1, which was recruited to a specific epigenetic landscape at Igk dictated by pre-B cell receptor (pre-BCR)-dependent Erk activation. BRWD1 enhanced RAG recruitment, increased gene accessibility and positioned nucleosomes 5' to each Jκ recombination signal sequence. BRWD1 thus targets recombination to Igk and places recombination within the context of signaling cascades that control B cell development. Our findings represent a paradigm in which, at any particular antigen-receptor locus, specialized mechanisms enforce lineage- and stage-specific recombination.

Molecular mechanisms insulating proliferation from genotoxic stress in B lymphocytes.

In mammals, B cells strictly segregate proliferation from somatic mutation as they develop within the bone marrow and then mature through germinal centers (GCs) in the periphery. Failure to do so risks autoimmunity and neoplastic transformation. Recent work has described how B cell progenitors transition between proliferation and mutation via cytokine signaling pathways, epigenetic chromatin regulation, and remodeling of 3D chromatin conformation. We propose a three-zone model of the GC that describes how proliferation and mutation are regulated. Using this model, we consider how recent mechanistic discoveries in B cell progenitors inform models of GC B cell function and reveal fundamental mechanisms underpinning humoral immunity, autoimmunity, and lymphomagenesis.

A self-reinforcing regulatory network triggered by limiting IL-7 activates pre-BCR signaling and differentiation.

The molecular crosstalk between the interleukin 7 receptor (IL-7R) and the precursor to the B cell antigen receptor (pre-BCR) in B lymphopoiesis has not been elucidated. Here we demonstrate that in pre-B cells, the IL-7R but not the pre-BCR was coupled to phosphatidylinositol-3-OH kinase (PI(3)K) and the kinase Akt; signaling by this pathway inhibited expression of recombination-activating gene 1 (Rag1) and Rag2. Attenuation of IL-7 signaling resulted in upregulation of the transcription factors Foxo1 and Pax5, which coactivated many pre-B cell genes, including Rag1, Rag2 and Blnk. Induction of Blnk (which encodes the signaling adaptor BLNK) enabled pre-BCR signaling via the signaling molecule Syk and promoted immunoglobulin light-chain rearrangement. BLNK expression also antagonized Akt activation, thereby augmenting the accumulation of Foxo1 and Pax5. This self-reinforcing molecular circuit seemed to sense limiting concentrations of IL-7 and functioned to constrain the proliferation of pre-B cells and trigger their differentiation.

Epigenetic repression of the Igk locus by STAT5-mediated recruitment of the histone methyltransferase Ezh2.

During B lymphopoiesis, recombination of the locus encoding the immunoglobulin κ-chain complex (Igk) requires expression of the precursor to the B cell antigen receptor (pre-BCR) and escape from signaling via the interleukin 7 receptor (IL-7R). By activating the transcription factor STAT5, IL-7R signaling maintains proliferation and represses Igk germline transcription by unknown mechanisms. We demonstrate that a STAT5 tetramer bound the Igk intronic enhancer (E(κi)), which led to recruitment of the histone methyltransferase Ezh2. Ezh2 marked trimethylation of histone H3 at Lys27 (H3K27me3) throughout the κ-chain joining region (J(κ)) to the κ-chain constant region (C(κ)). In the absence of Ezh2, IL-7 failed to repress Igk germline transcription. H3K27me3 modifications were lost after termination of IL-7R-STAT5 signaling, and the transcription factor E2A bound E(κi), which resulted in acquisition of H3K4me1 and acetylated histone H4 (H4Ac). Genome-wide analyses showed a STAT5 tetrameric binding motif associated with transcriptional repression. Our data demonstrate how IL-7R signaling represses Igk germline transcription and provide a general model for STAT5-mediated epigenetic transcriptional repression.

Artificial Intelligence and Cellular Segmentation in Tissue Microscopy Images.

With applications in object detection, image feature extraction, image classification, and image segmentation, artificial intelligence is facilitating high-throughput analysis of image data in a variety of biomedical imaging disciplines, ranging from radiology and pathology to cancer biology and immunology. Specifically, a growth in research on deep learning has led to the widespread application of computer-visualization techniques for analyzing and mining data from biomedical images. The availability of open-source software packages and the development of novel, trainable deep neural network architectures has led to increased accuracy in cell detection and segmentation algorithms. By automating cell segmentation, it is now possible to mine quantifiable cellular and spatio-cellular features from microscopy images, providing insight into the organization of cells in various pathologies. This mini-review provides an overview of the current state of the art in deep learning- and artificial intelligence-based methods of segmentation and data mining of cells in microscopy images of tissue.

Cellular aspects of the pathogenesis of lupus nephritis.

PURPOSE OF REVIEW: Lupus nephritis is a common severe manifestation of systemic lupus erythematosus. Despite recent advances in therapeutics and understanding of its pathogenesis, there are still substantial unmet needs. This review discusses recent discoveries in these areas, especially the role of tubulointerstitial inflammation (TII) in lupus nephritis. RECENT FINDINGS: Non-white ethnicity is still a major risk and poor prognostic factor in lupus nephritis. TII and fibrosis have been found to be associated with worse renal outcome but the current lupus nephritis treatment guidelines and trials are based on the degree of glomerular inflammation. In combination with mycophenolate mofetil, a B-cell-targeted therapy (belimumab) and a calcineurin inhibitor (voclosporin) have shown efficacy in recent lupus nephritis trials. However, response rates have been modest. While lupus glomerulonephritis results from immune complex deposition derived from systemic autoantibodies, TII arises from complex processes associated with in situ adaptive cell networks. These include local antibody production, and cognate or antigen-induced interactions between T follicular helper cells, and likely other T-cell populations, with antigen presenting cells including B cells, myeloid dendritic cells and plasmacytoid dendritic cells. SUMMARY: Better understanding of the pathogenesis of TII will identify novel therapeutic targets predicted to improve outcomes in our patients with lupus nephritis.

Ras orchestrates exit from the cell cycle and light-chain recombination during early B cell development.

Signals through the pre-B cell antigen receptor (pre-BCR) and interleukin 7 receptor (IL-7R) coordinate pre-B cell population expansion with subsequent recombination of the locus encoding immunoglobulin kappa-chain (Igk). Although many 'downstream' effectors of each receptor are known, how they integrate to mediate development has remained unclear. Here we report that pre-BCR-mediated activation of the Ras-MEK-Erk signaling pathway silenced transcription of Ccnd3 (encoding cyclin D3) and coordinated exit from the cell cycle with induction of the transcription factor E2A and the initiation of Igk recombination. IL-7R-mediated activation of the transcription factor STAT5 opposed this pathway by promoting Ccnd3 expression and concomitantly inhibiting Igk transcription by binding to the Igk intronic enhancer and preventing E2A recruitment. Our data show how pre-BCR signaling poises pre-B cells to undergo differentiation after escape from IL-7R signaling.

Antibodies in cerebral cavernous malformations react with cytoskeleton autoantigens in the lesional milieu.

Previous studies have reported robust inflammatory cell infiltration, synthesis of IgG, B-cell clonal expansion, deposition of immune complexes and complement within cerebral cavernous malformation (CCM) lesions. B-cell depletion has also been shown to reduce the maturation of CCM in murine models. We hypothesize that antigen(s) within the lesional milieu perpetuate the pathogenetic immune responses in CCMs. This study aims to identify those putative antigen(s) using monoclonal antibodies (mAbs) derived from plasma cells found in surgically removed human CCM lesions. We produced human mAbs from laser capture micro-dissected plasma cells from four CCM patients, and also germline-reverted versions. CCM mAbs were assayed using immunofluorescence on central nervous system (CNS) tissues and immunocytochemistry on human primary cell lines. Antigen characterization was performed using a combination of confocal microscopy, immunoprecipitation and mass spectrometry. Affinity was determined by enzyme-linked immunosorbent assay, and specificity by multi-color confocal microscopy and quantitative co-localization. CCM mAbs bound CNS tissue, especially endothelial cells and astrocytes. Non-muscle myosin heavy chain IIA (NMMHCIIA), vimentin and tubulin are three cytoskeleton proteins that were commonly targeted. Selection of cytoskeleton proteins by plasma cells was supported by a high frequency of immunoglobulin variable region somatic hypermutations, high affinity and selectivity of mAbs in their affinity matured forms, and profoundly reduced affinity and selectivity in the germline reverted forms. Antibodies produced by plasma cells in CCM lesions commonly target cytoplasmic and cytoskeletal autoantigens including NMMHCIIA, vimentin and tubulin that are abundant in endothelial cells and astrocytes. Binding to, and selection on autoantigen(s) in the lesional milieu likely perpetuates the pathogenetic immune response in CCMs. Blocking this in situ autoimmune response may yield a novel treatment for CCM.

EZH2 Regulates the Developmental Timing of Effectors of the Pre-Antigen Receptor Checkpoints.

The histone methyltransferase EZH2 is required for B and T cell development; however, the molecular mechanisms underlying this requirement remain elusive. In a murine model of lymphoid-specific EZH2 deficiency we found that EZH2 was required for proper development of adaptive, but not innate, lymphoid cells. In adaptive lymphoid cells EZH2 prevented the premature expression of Cdkn2a and the consequent stabilization of p53, an effector of the pre-Ag receptor checkpoints. Deletion of Cdkn2a in EZH2-deficient lymphocytes prevented p53 stabilization, extended lymphocyte survival, and restored differentiation resulting in the generation of mature B and T lymphocytes. Our results uncover a crucial role for EZH2 in adaptive lymphocytes to control the developmental timing of effectors of the pre-Ag receptor checkpoints.