Pregnancy dedifferentiates memory CD8+ T cells into hypofunctional cells with exhaustion-enriched programs.

Alloreactive memory, unlike naive, CD8+ T cells resist transplantation tolerance protocols and are a critical barrier to long-term graft acceptance in the clinic. We here show that semiallogeneic pregnancy successfully reprogrammed memory fetus/graft-specific CD8+ T cells (TFGS) toward hypofunction. Female C57BL/6 mice harboring memory CD8+ T cells generated by the rejection of BALB/c skin grafts and then mated with BALB/c males achieved rates of pregnancy comparable with naive controls. Postpartum CD8+ TFGS from skin-sensitized dams upregulated expression of T cell exhaustion (TEX) markers (Tox, Eomes, PD-1, TIGIT, and Lag3). Transcriptional analysis corroborated an enrichment of canonical TEX genes in postpartum memory TFGS and revealed a downregulation of a subset of memory-associated transcripts. Strikingly, pregnancy induced extensive epigenetic modifications of exhaustion- and memory-associated genes in memory TFGS, whereas minimal epigenetic modifications were observed in naive TFGS. Finally, postpartum memory TFGS durably expressed the exhaustion-enriched phenotype, and their susceptibility to transplantation tolerance was significantly restored compared with memory TFGS. These findings advance the concept of pregnancy as an epigenetic modulator inducing hypofunction in memory CD8+ T cells that has relevance not only for pregnancy and transplantation tolerance, but also for tumor immunity and chronic infections.

BRWD1 establishes epigenetic states for germinal center initiation, maintenance, and function.

Germinal center (GC) B cells segregate into three subsets that compartmentalize the antagonistic molecular programs of selection, proliferation, and somatic hypermutation. In bone marrow, the epigenetic reader BRWD1 orchestrates and insulates the sequential stages of cell proliferation and Igk recombination. We hypothesized BRWD1 might play similar insulative roles in the periphery. In Brwd1 -/- follicular B cells, GC initiation and class switch recombination following immunization were inhibited. In contrast, in Brwd1 -/- GC B cells there was admixing of chromatin accessibility across GC subsets and transcriptional dysregulation including induction of inflammatory pathways. This global molecular GC dysregulation was associated with specific defects in proliferation, affinity maturation, and tolerance. These data suggest that GC subset identity is required for some but not all GC-attributed functions. Furthermore, these data demonstrate a central role for BRWD1 in orchestrating epigenetic transitions at multiple steps along B cell developmental and activation pathways.

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.

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.

STAT3 signaling in B cells controls germinal center zone organization and recycling.

Germinal centers (GCs), sites of antibody affinity maturation, are organized into dark (DZ) and light (LZ) zones. Here, we show a B cell-intrinsic role for signal transducer and activator of transcription 3 (STAT3) in GC DZ and LZ organization. Altered zonal organization of STAT3-deficient GCs dampens development of long-lived plasma cells (LL-PCs) but increases memory B cells (MBCs). In an abundant antigenic environment, achieved here by prime-boost immunization, STAT3 is not required for GC initiation, maintenance, or proliferation but is important for sustaining GC zonal organization by regulating GC B cell recycling. Th cell-derived signals drive STAT3 tyrosine 705 and serine 727 phosphorylation in LZ B cells, regulating their recycling into the DZ. RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) analyses identified STAT3 regulated genes that are critical for LZ cell recycling and transiting through DZ proliferation and differentiation phases. Thus, STAT3 signaling in B cells controls GC zone organization and recycling, and GC egress of PCs, but negatively regulates MBC output.

Pregnancy programs epigenetic and transcriptional exhaustion in memory CD8+ T cells.

Alloreactive memory T cells, unlike naive T cells, fail to be restrained by transplantation tolerance protocols or regulatory T cells, and therefore represent a critical barrier to long-term graft acceptance. Using female mice sensitized by rejection of fully-mismatched paternal skin allografts, we show that subsequent semi-allogeneic pregnancy successfully reprograms memory fetus/graft-specific CD8+ T cells (TFGS) towards hypofunction in a manner that is mechanistically distinct from naive TFGS. Post-partum memory TFGS were durably hypofunctional, exhibiting enhanced susceptibility to transplantation tolerance induction. Furthermore, multi-omics studies revealed that pregnancy induced extensive phenotypic and transcriptional modifications in memory TFGS reminiscent of T cell exhaustion. Strikingly, at loci transcriptionally modified in both naive and memory TFGS during pregnancy, chromatin remodeling was observed exclusively in memory and not naive TFGS. These data reveal a novel link between T cell memory and hypofunction via exhaustion circuits and pregnancy-mediated epigenetic imprinting. This conceptual advance has immediate clinical relevance to pregnancy and transplantation tolerance.

Asymmetrical forward and reverse developmental trajectories determine molecular programs of B cell antigen receptor editing.

During B lymphopoiesis, B cell progenitors progress through alternating and mutually exclusive stages of clonal expansion and immunoglobulin (Ig) gene rearrangements. Great diversity is generated through the stochastic recombination of Ig gene segments encoding heavy and light chain variable domains. However, this commonly generates autoreactivity. Receptor editing is the predominant tolerance mechanism for self-reactive B cells in the bone marrow (BM). B cell receptor editing rescues autoreactive B cells from negative selection through renewed light chain recombination first at Igκ then Igλ loci. Receptor editing depends on BM microenvironment cues and key transcription factors such as NF-κB, FOXO, and E2A. The specific BM factor required for receptor editing is unknown. Furthermore, how transcription factors coordinate these developmental programs to promote usage of the λ chain remains poorly defined. Therefore, we used two mouse models that recapitulate pathways by which Igλ light chain-positive B cells develop. The first has deleted J kappa (Jκ) genes and hence models Igλ expression resulting from failed Igκ recombination (Igκdel). The second models autoreactivity by ubiquitous expression of a single-chain chimeric anti-Igκ antibody (κ-mac). Here, we demonstrated that autoreactive B cells transit asymmetric forward and reverse developmental trajectories. This imparted a unique epigenetic landscape on small pre-B cells, which opened chromatin to transcription factors essential for Igλ recombination. The consequences of this asymmetric developmental path were both amplified and complemented by CXCR4 signaling. These findings reveal how intrinsic molecular programs integrate with extrinsic signals to drive receptor editing.

Control of Early B Cell Development by the RNA N6-Methyladenosine Methylation.

The RNA N6-methyladenosine (m6A) methylation is installed by the METTL3-METTL14 methyltransferase complex. This modification has critical regulatory roles in various biological processes. Here, we report that deletion of Mettl14 dramatically reduces mRNA m6A methylation in developing B cells and severely blocks B cell development in mice. Deletion of Mettl14 impairs interleukin-7 (IL-7)-induced pro-B cell proliferation and the large-pre-B-to-small-pre-B transition and causes dramatic abnormalities in gene expression programs important for B cell development. Suppression of a group of transcripts by cytoplasmic m6A reader YTHDF2 is critical to the IL-7-induced pro-B cell proliferation. In contrast, the block in the large-pre-B-to-small-pre-B transition is independent of YTHDF1 or YTHDF2 but is associated with a failure to properly upregulate key transcription factors regulating this transition. Our data highlight the important regulatory roles of the RNA m6A methylation and its reader proteins in early B cell development.

It Takes Three Receptors to Raise a B Cell.

As the unique source of diverse immunoglobulin repertoires, B lymphocytes are an indispensable part of humoral immunity. B cell progenitors progress through sequential and mutually exclusive states of proliferation and recombination, coordinated by cytokines and chemokines. Mutations affecting the crucial pre-B cell checkpoint result in immunodeficiency, autoimmunity, and leukemia. This checkpoint was previously modeled by the signaling of two opposing receptors, IL-7R and the pre-BCR. We provide an update to this model in which three receptors, IL-7R, pre-BCR, and CXCR4, work in concert to coordinate both the proper positioning of B cell progenitors in the bone marrow (BM) microenvironment and their progression through the pre-B checkpoint. Furthermore, signaling initiated by all three receptors directly instructs cell fate and developmental progression.

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.

B-1a cells acquire their unique characteristics by bypassing the pre-BCR selection stage.

B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the peritoneal and pleural cavities. In contrast to conventional B-2 cells, B-1a cells have a receptor repertoire that is biased towards bacterial and self-antigens, promoting a rapid response to infection and clearing of apoptotic cells. Although B-1a cells are known to primarily originate from fetal tissues, the mechanisms by which they arise has been a topic of debate for many years. Here we show that in the fetal liver versus bone marrow environment, reduced IL-7R/STAT5 levels promote immunoglobulin kappa gene recombination at the early pro-B cell stage. As a result, differentiating B cells can directly generate a mature B cell receptor (BCR) and bypass the requirement for a pre-BCR and pairing with surrogate light chain. This 'alternate pathway' of development enables the production of B cells with self-reactive, skewed specificity receptors that are peculiar to the B-1a compartment. Together our findings connect seemingly opposing lineage and selection models of B-1a cell development and explain how these cells acquire their unique properties.

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.

Monitoring Changes of Cardio-Respiratory Parameters During 2000m Rowing Performance.

The purpose of this study was to characterize the kinetics of cardio-respiratory parameters of elite male rowers during 2000m rowing time trial. 16 lightweight category (LWC) and 11 open category (OC) elite male rowers attending National camp were included in the study. Pulmonary gas exchange and heart rate (HR) during 2000m rowing ergometer test was determined through breath-by-breath analysis, with a portable metabolic gas analyzer and HR monitor. Time to completion, HR, oxygen uptake (V̇O2), minute ventilation (V̇E) and respiratory exchange ratio (RER) were recorded at 500m, 1000m, 1500m and 2000m intervals. No significant (p>0.05) difference was observed in the HR kinetics during 2000m rowing between the groups. However, split HR during the entire course was on the higher side in OC than LWC. Relative V̇O2 at 1000m (p<0.01), 1500m (p<0.05) and 2000m (p<0.01) was significantly less in OC rowers compared to LWC. However, V̇E was significantly higher for the OC group at 1500m (p<0.05) and 2000m (p<0.01) whereas RER was only significantly higher at 2000m (p<0.05). %change in absolute and relative V̇O2, V̇E and RER at each 500m interval showed no significant difference among the groups. OC rowers had taken significantly less time (p<0.05) to complete first 500m, 500m to 1000m and last 500m distance than LWC rowers. This detailed insight of rower's physiological responses can help coaches and support staff to determine the physiological working capacity of rowers at different levels, predicting performance and provided normative ranges for developing a representative physiological profile of elite Indian rowers.

BRWD1 orchestrates epigenetic landscape of late B lymphopoiesis.

Transcription factor (TF) networks determine cell fate in hematopoiesis. However, how TFs cooperate with other regulatory mechanisms to instruct transcription remains poorly understood. Here we show that in small pre-B cells, the lineage restricted epigenetic reader BRWD1 closes early development enhancers and opens the enhancers of late B lymphopoiesis to TF binding. BRWD1 regulates over 7000 genes to repress proliferative and induce differentiation programs. However, BRWD1 does not regulate the expression of TFs required for B lymphopoiesis. Hypogammaglobulinemia patients with BRWD1 mutations have B-cell transcriptional profiles and enhancer landscapes similar to those observed in Brwd1-/- mice. These data indicate that, in both mice and humans, BRWD1 is a master orchestrator of enhancer accessibility that cooperates with TF networks to drive late B-cell development.

Regulated Capture of Vκ Gene Topologically Associating Domains by Transcription Factories.

Expression of vast repertoires of antigen receptors by lymphocytes, with each cell expressing a single receptor, requires stochastic activation of individual variable (V) genes for transcription and recombination. How this occurs remains unknown. Using single-cell RNA sequencing (scRNA-seq) and allelic variation, we show that individual pre-B cells monoallelically transcribe divergent arrays of Vκ genes, thereby opening stochastic repertoires for subsequent Vκ-Jκ recombination. Transcription occurs upon translocation of Vκ genes to RNA polymerase II arrayed on the nuclear matrix in transcription factories. Transcription is anchored by CTCF-bound sites or E2A-loaded Vκ promotors and continues over large genomic distances delimited only by topological associating domains (TADs). Prior to their monoallelic activation, Vκ loci are transcriptionally repressed by cyclin D3, which prevents capture of Vκ gene containing TADs by transcription factories. Cyclin D3 also represses protocadherin, olfactory, and other monoallelically expressed genes, suggesting a widely deployed mechanism for coupling monoallelic gene activation with cell cycle exit.

Publisher Correction: A shared Runx1-bound Zbtb16 enhancer directs innate and innate-like lymphoid lineage development.

In the original PDF version of this Article, which was published on 16 October 2017, the publication date was incorrectly given as 11 October 2017. This has now been corrected in the PDF; the HTML version of the paper was correct from the time of publication.

Igß ubiquitination activates PI3K signals required for endosomal sorting.

A wealth of in vitro data has demonstrated a central role for receptor ubiquitination in endocytic sorting. However, how receptor ubiquitination functions in vivo is poorly understood. Herein, we report that ablation of B cell antigen receptor ubiquitination in vivo uncouples the receptor from CD19 phosphorylation and phosphatidylinositol 3-kinase (PI3K) signals. These signals are necessary and sufficient for accumulating phosphatidylinositol (3,4,5)-trisphosphate (PIP3) on B cell receptor-containing early endosomes and proper sorting into the MHC class II antigen-presenting compartment (MIIC). Surprisingly, MIIC targeting is dispensable for T cell-dependent immunity. Rather, it is critical for activating endosomal toll-like receptors and antiviral humoral immunity. These findings demonstrate a novel mechanism of receptor endosomal signaling required for specific peripheral immune responses.

A shared Runx1-bound Zbtb16 enhancer directs innate and innate-like lymphoid lineage development.

Zbtb16-encoded PLZF is a signature transcription factor (TF) that directs the acquisition of T-helper effector programs during the development of multiple innate lymphocyte lineages, including natural killer T cell, innate lymphoid cell, mucosal-associated invariant T cell and γδ lineages. PLZF is also essential in osteoblast and spermatogonial development. How Zbtb16 itself is regulated in different lineages is incompletely understood. Here, by systematic CRISPR/Cas9-assisted deletions of chromatin accessible regions within the Zbtb16 locus in mouse, we identify a critical enhancer controlling PLZF expression exclusively in innate lymphoid lineages. Multiple sites within this enhancer express canonical motifs for the TF Runx1, which is essential for the development of these lineages. Notably, some regulatory sites control the kinetic rather than the overall level of PLZF expression. Thus, our comprehensive, unbiased analysis of regulatory elements in vivo reveals critical mechanisms of Zbtb16 regulation shared between innate and innate-like lymphoid lineages. Zbtb16-encoded transcription factor PLZF directs the differentiation of multiple innate and innate-like cell lineages, but how Zbtb16 itself is regulated remains unclear. Here the authors show, using CRISPR gene editing, ATAC-seq and ChIP-seq, that specific Runx1-bound enhancer elements critically modulate lineage-dependent expressions of PLZF.

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.

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.