Temporal dynamics and genomic programming of plasma cell fates.

Affinity-matured plasma cells (PCs) of varying lifespans are generated through a germinal center (GC) response. The developmental dynamics and genomic programs of antigen-specific PC precursors remain to be elucidated. Here, using a model antigen in mice, we demonstrate biphasic generation of PC precursors, with those generating long-lived bone marrow PCs preferentially produced in the late phase of GC response. Clonal tracing using single-cell RNA sequencing and B cell antigen receptor sequencing in spleen and bone marrow compartments, coupled with adoptive transfer experiments, reveals a new PC transition state that gives rise to functionally competent PC precursors. The latter undergo clonal expansion, dependent on inducible expression of TIGIT. We propose a model for the proliferation and programming of precursors of long-lived PCs, based on extended antigen encounters in the GC.

Temporal dynamics and genomic programming of plasma cell fates.

Affinity-matured plasma cells (PCs) of varying lifespans are generated through a germinal center (GC) response. The developmental dynamics and genomic programs of antigen-specific PC precursors remain to be elucidated. Using a model antigen, we demonstrate biphasic generation of PC precursors, with those generating long-lived bone marrow PCs preferentially produced in the late phase of GC response. Clonal tracing using scRNA-seq+BCR-seq in spleen and bone marrow compartments, coupled with adoptive transfer experiments, reveal a novel PC transition state that gives rise to functionally competent PC precursors. The latter undergo clonal expansion, dependent on inducible expression of TIGIT. We propose a model for the proliferation and programming of precursors of long-lived PCs, based on extended antigen encounters followed by reduced antigen availability.

Comprehensive analyses of B-cell compartments across the human body reveal novel subsets and a gut-resident memory phenotype.

Although human B cells have been extensively studied, most reports have used peripheral blood as a source. Here, we used a unique tissue resource derived from healthy organ donors to deeply characterize human B-cell compartments across multiple tissues and donors. These datasets revealed that B cells in the blood are not in homeostasis with compartments in other tissues. We found striking donor-to-donor variability in the frequencies and isotype of CD27+ memory B cells (MBCs). A comprehensive antibody-based screen revealed markers of MBC and allowed identification of novel MBC subsets with distinct functions defined according to surface expression of CD69 and CD45RB. We defined a tissue-resident MBC phenotype that was predominant in the gut but absent in blood. RNA-sequencing of MBC subsets from multiple tissues revealed a tissue-resident MBC gene signature as well as gut- and spleen-specific signatures. Overall, these studies provide novel insights into the nature and function of human B-cell compartments across multiple tissues.

CD91 on dendritic cells governs immunosurveillance of nascent, emerging tumors.

The immune system detects aberrant, premalignant cells and eliminates them before the development of cancer. Immune cells, including T cells, have been shown to be critical components in eradicating these aberrant cells, and when absent in the host, incidence of cancer increases. Here, we show that CD91, a receptor expressed on antigen-presenting cells, is required for priming immune responses to nascent, emerging tumors. In the absence of CD91, effector immune responses are subdued, and tumor incidence and progression are amplified. We also show that, consequently, tumors that arise in the absence of CD91 express neo-epitopes with indices that are indicative of greater immunogenicity. Polymorphisms in human CD91 that are expected to affect ligand binding are shown to influence antitumor immune responses in cancer patients. This study presents a molecular mechanism for priming immune responses to nascent, emerging tumors that becomes a predictor of cancer susceptibility and progression.

Bone marrow lympho-myeloid malfunction in obesity requires precursor cell-autonomous TLR4.

Obesity, a prevalent condition in adults and children, impairs bone marrow (BM) function. However, the underlying mechanisms are unclear. Here, we show that obese mice exhibit poor emergency immune responses in a toll-like receptor 4 (TLR4)-dependent manner. Canonical myeloid genes (Csf1r, Spi1, Runx1) are enhanced, and lymphoid genes (Flt3, Tcf3, Ebf1) are reduced. Using adoptive transfer and mixed BM chimera approaches we demonstrate that myeloid>lymphoid bias arises after 6 weeks of high-fat diet and depends on precursor cell-autonomous TLR4. Further, lean mice exposed to the TLR4 ligand lipopolysaccharide (LPS) at doses similar to that detectable in obese serum recapitulates BM lympho-myeloid alterations. Together, these results establish a mechanistic contribution of BM cell-intrinsic TLR4 to obesity-driven BM malfunction and demonstrate the importance of LPS. Our findings raises important questions about the impact of maternal obesity and endotoxemia to fetal hematopoiesis, as fetal immune precursors are also sensitive to TLR4 signals.

Sex-, stress-, and sympathetic post-ganglionic-dependent changes in identity and proportions of immune cells in the dura.

Aim of investigation Due to compelling evidence in support of links between sex, stress, sympathetic post-ganglionic innervation, dural immune cells, and migraine, our aim was to characterize the impacts of these factors on the type and proportion of immune cells in the dura. Methods Dural immune cells were obtained from naïve or stressed adult male and female Sprague Dawley rats for flow cytometry. Rats with surgical denervation of sympathetic post-ganglionic neurons of the dura were also studied. Results Immune cells comprise ∼17% of all cells in the dura. These included: macrophages/granulocytes ("Macs"; 63.2% of immune cells), dendritic cells (0.88%), T-cells (4.51%), natural killer T-cells (0.51%), natural killer cells (3.08%), and B-cells (20.0%). There were significantly more Macs and fewer B- and natural killer T-cells in the dura of females compared with males. Macs and dendritic cells were significantly increased by stress in males, but not females. In contrast, T-cells were significantly increased in females with a 24-hour delay following stress. Lastly, Macs, dendritic cells, and T-cells were significantly higher in sympathectomized-naïve males, but not females. Conclusions It may not only be possible, but necessary to use different strategies for the most effective treatment of migraine in men and women.

Cutting Edge: Hematopoietic Stem Cell Expansion and Common Lymphoid Progenitor Depletion Require Hematopoietic-Derived, Cell-Autonomous TLR4 in a Model of Chronic Endotoxin.

Hematopoietic stem and progenitors cells (HSPCs) are activated through TLR4 in vitro. However, it remains unclear whether in vivo TLR4 sensing by HSPCs occurs directly or via other cell intermediates. In this study, we examined the cellular mechanisms underlying murine hematopoietic stem cell (HSC) expansion and common lymphoid progenitor (CLP) depletion in a model of chronic low-dose LPS. Using adoptive-transfer approaches, we show that HSC and CLP sensitivity to chronic LPS depends on hematopoietic-derived, cell subset-autonomous TLR4. Like murine progenitors, human HSPCs are activated by TLR4 in vitro. Using humanized mice, a preclinical model relevant to human physiology, we show that persistent endotoxin increases the frequency of Ki-67(+) HSCs and severely depletes CLPs and B precursors. Together, our findings show that murine HSPCs directly respond to endotoxin in vivo and that persistent LPS, a feature of several diseases of global health significance, impairs human lymphopoiesis.

Sex-, Stress-, and Sympathetic Post-Ganglionic Neuron-Dependent Changes in the Expression of Pro- and Anti-Inflammatory Mediators in Rat Dural Immune Cells.

BACKGROUND: Migraine attacks are associated with sterile inflammation of the dura. Immune cells are a primary source of inflammatory mediators, and we therefore sought to further explore the link between dural immune cells and migraine. OBJECTIVE: Based on the observations that migraine is more common in women than in men, stress is the most common trigger for a migraine attack, and sympathetic post-ganglionic innervation of the dura enables local control of dural immune cells, we hypothesized that stress shifts the balance of inflammatory mediator expression in dural immune cells toward those that trigger a migraine attack, where these changes are larger in females and dependent, at least in part, on sympathetic post-ganglionic innervation of the dura. Our objective was to test this hypothesis. METHODS: Dura were obtained from naïve or stressed, intact or surgically sympathectomized, adult male and female rats. Dura were assessed immediately or 24 hours after termination of 4 continuous days of unpredictable, mild stressors. Following enzymatic digestion of each dura, myeloid and lymphoid-derived dural immune cells were isolated by fluorescence-activated cell sorting for semi-quantitative polymerase chain reaction analysis. RESULTS: In myeloid-derived dural immune cells, there was an increase in pro-inflammatory mediator mRNA following stress, particularly in females, which remained elevated with a 24-hour delay after stress. There was a stress-induced decrease in anti-inflammatory mediator mRNA immediately after stress in females, but not males. The stress-induced changes were attenuated in sympathectomized females. In lymphoid-derived dural immune cells, there was a persistent increase in pro-inflammatory mediator mRNA following stress, particularly in females. A stress-induced increase in anti-inflammatory mediator mRNA was also observed in both males and females, and was further attenuated in sympathectomized females. CONCLUSIONS: Consistent with our hypothesis, there is a stress-induced shift in the balance of pro- and anti-inflammatory mediator expression in dural immune cells that is more pronounced in females, and is dependent, at least in part, on sympathetic post-ganglionic innervation in females. This shift in the balance of inflammatory mediator expression may not only play an important role in triggering migraine attacks, but also suggests it may be possible, if not necessary, to employ different strategies to most effectively treat migraine in men and women.

Transcription elongation factor ELL2 drives Ig secretory-specific mRNA production and the unfolded protein response.

Differentiation of B cells into Ab-secreting cells induces changes in gene transcription, IgH RNA processing, the unfolded protein response (UPR), and cell architecture. The transcription elongation factor eleven nineteen lysine-rich leukemia gene (ELL2) stimulates the processing of the secreted form of the IgH mRNA from the H chain gene. Mice (mus musculus) with the ELL2 gene floxed in either exon 1 or exon 3 were constructed and crossed to CD19-driven cre/CD19(+). The B cell-specific ELL2 conditional knockouts (cKOs; ell2(loxp/loxp) CD19(cre/+)) exhibit curtailed humoral responses both in 4-hydroxy-3-nitrophenyl acetyl-Ficoll and in 4-hydroxy-3-nitrophenyl acetyl-keyhole limpet hemocyanin immunized animals; recall responses were also diminished. The number of immature and recirculating B cells in the bone marrow is increased in the cKOs, whereas plasma cells in spleen are reduced relative to control animals. There are fewer IgG1 Ab-producing cells in the bone marrow of cKOs. LPS ex vivo-stimulated B220(lo)CD138(+) cells from ELL2-deficient mouse spleens are 4-fold less abundant than from control splenic B cells; have a paucity of secreted IgH; and have distended, abnormal-appearing endoplasmic reticulum. IRE1α is efficiently phosphorylated, but the amounts of Ig κ, ATF6, BiP, Cyclin B2, OcaB (BOB1, Pou2af1), and XBP1 mRNAs, unspliced and spliced, are severely reduced in ELL2-deficient cells. ELL2 enhances the expression of BCMA (also known as Tnfrsf17), which is important for long-term survival. Transcription yields from the cyclin B2 and the canonical UPR promoter elements are upregulated by ELL2 cDNA. Thus, ELL2 is important for many aspects of Ab secretion, XBP1 expression, and the UPR.

Hematopoiesis in steady-state versus stress: self-renewal, lineage fate choice, and the conversion of danger signals into cytokine signals in hematopoietic stem cells.

Long-term hematopoietic stem cells (LT-HSCs) replenish the innate and adaptive immune compartments throughout life. Although significant progress has defined the major transcription factors that regulate lineage specification, the architectural proteins that globally coordinate DNA methylation, histone modification, and changes in gene expression are poorly defined. Provocative new studies establish the chromatin organizer special AT-rich binding protein 1 (Satb1) as one such global regulator in LT-HSCs. Satb1 is a nuclear organizer that partitions chromatin through the formation of cage-like structures. By integrating epigenetic and transcriptional pathways, Satb1 coordinates LT-HSC division, self-renewal, and lymphoid potential. Unexpected among the assortment of genes under Satb1 control in hematopoietic stem cells (HSCs) are cytokines, a finding that takes on additional importance with the provocative finding that short-term HSCs and downstream multipotent progenitors are potent and biologically relevant cytokine secretors during stress-mediated hematopoiesis. Together, these studies reveal a new mechanism of fate regulation and an unforeseen functional capability of HSCs.

Cell-intrinsic in vivo requirement for the E47-p21 pathway in long-term hematopoietic stem cells.

Major regulators of long-term hematopoietic stem cell (LT-HSC) self-renewal and proliferation have been identified, but knowledge of their in vivo interaction in a linear pathway is lacking. In this study, we show a direct genetic link between the transcription factor E47 and the major cell cycle regulator p21 in controlling LT-HSC integrity in vivo under repopulation stress. Numerous studies have shown that E47 activates p21 transcription in hematopoietic subsets in vitro, and we now reveal the in vivo relevance of the E47-p21 pathway by reducing the gene dose of each factor individually (E47(het) or p21(het)) versus in tandem (E47(het)p21(het)). E47(het)p21(het) LT-HSCs and downstream short-term hematopoietic stem cells exhibit hyperproliferation and preferential susceptibility to mitotoxin compared to wild-type or single haploinsufficient controls. In serial adoptive transfers that rigorously challenge self-renewal, E47(het)p21(het) LT-HSCs dramatically and progressively decline, indicating the importance of cell-intrinsic E47-p21 in preserving LT-HSCs under stress. Transient numeric recovery of downstream short-term hematopoietic stem cells enabled the production of functionally competent myeloid but not lymphoid cells, as common lymphoid progenitors were decreased, and peripheral lymphocytes were virtually ablated. Thus, we demonstrate a developmental compartment-specific and lineage-specific requirement for the E47-p21 pathway in maintaining LT-HSCs, B cells, and T cells under hematopoietic repopulation stress in vivo.

Hematopoiesis sculpted by pathogens: Toll-like receptors and inflammatory mediators directly activate stem cells.

Hematopoietic stem cells (HSCs) repopulate the immune system during normal replenishment as well as under the burden of pathogen stress, but the respective outcomes of differentiation are not the same. Under homeostatic conditions such as those which accompany turnover of immune cell subsets, HSCs appear to co-equally prime genes associated with the major downstream lineages: lymphoid, myeloid, and megakaryocyte/erythroid. Recent studies reveal, however, that during pathogen exposure, hematopoiesis may yield progeny in proportions different than those produced under homeostasis. At least some of these effects may be due to pathogen engagement of Toll-like receptors (TLRs) expressed on HSCs. HSCs are also responsive to inflammatory cytokines that are produced in response to pathogen burden and are present in the bone marrow microenvironment. Thus, hematopoiesis is not a formulaic process that produces the same, predictable outcome regardless of the specific environmental context. Rather, hematopoiesis represents a dynamic biological system that can be appreciably responsive to environmental factors, an influence that extends to the level of the HSC itself. Knowledge of functional consequences of TLR ligation on HSCs may be therapeutically exploited and applied to treatment of hematopoietic insufficiency in the setting of infection and disease.

Transcriptional and epigenetic regulation of B cell development.

B cell development starts in the bone marrow where hematopoietic stem cells (HSCs) progress through sequential developmental stages, as it differentiates into a naïve B cell expressing surface immunoglobulin. In the periphery, B cells that encounter antigen can further differentiate into antibody-secreting plasma cells. In this review, we focus on two factors, E47 and ELL2, which play important roles in the regulation of B cell development in the bone marrow and differentiation of mature B cells into plasma cells in the periphery, respectively. First, E47 activity is required for B cell development in the bone marrow. In addition, we have identified a cell-intrinsic role for E47 in regulating efficient self-renewal and long-term multilineage bone marrow reconstitution potential of HSCs. Second, we explored the role of transcription elongation factors in the super elongation complex (SEC), including ELL2 (eleven-nineteen lysine-rich leukemia factor) in driving poly(A) site choice and plasma cell development. We found that elongation factors impel high levels of IgH mRNA production and alternative processing at the promoter proximal, secretory-specific (sec) poly(A) site in plasma cells by enhancing RNA polymerase II modifications and downstream events. The sec poly(A) site, essentially hidden in B cells, is found by SEC factors in plasma cells.

The B lineage transcription factor E2A regulates apoptosis in chronic lymphocytic leukemia (CLL) cells.

Chronic lymphocytic leukemia (CLL) is a common malignancy characterized by the accumulation of B lymphocytes with an antigen-experienced activated CD19(+)CD5(+) clonal phenotype. Clinically, ∼50% of cases will behave more aggressively. Here, we investigate the role of the major B-cell transcription factor E2A, a known regulator of B-cell survival and proliferation, to CLL persistence. We show that E2A is elevated at the mRNA and protein levels relative to normal B-cell subsets. E2A silencing in primary CLL cells leads to a significant increase in spontaneous apoptosis in both CD38(+) (aggressive) and CD38(-) (indolent) cases. Moreover, E2A knockdown synergizes with the immunomodulatory drug lenalidomide to reduce CLL viability. E2A is known to restrain the proliferation of primary B and T lymphocytes at multiple stages of maturation and we report that targeted E2A disruption increases the frequency of Ki-67(+) CLL cells in the absence of effects on de novo proliferation. At the molecular level, E2A siRNA-treated CLL cells display reduced expression of key genes associated with survival and cell cycling including p27, p21 and mcl-1, of which the former two are known E2A target genes. Thus, E2A, a key transcription factor associated with the B-cell activation profile, regulates apoptosis in CLL and may contribute to disease pathology.

Spermatogenetic but not immunological defects in mice lacking the τCstF-64 polyadenylation protein.

Alternative polyadenylation controls expression of genes in many tissues including immune cells and male germ cells. The τCstF-64 polyadenylation protein is expressed in both cell types, and we previously showed that Cstf2t, the gene encoding τCstF-64 was necessary for spermatogenesis and fertilization. Here we examine consequences of τCstF-64 loss in both germ cells and immune cells. Spermatozoa from Cstf2t null mutant (Cstf2t(-/-)) mice of ages ranging from 60 to 108 days postpartum exhibited severe defects in motility and morphology that were correlated with a decrease in numbers of round spermatids. Spermatozoa in these mice also displayed severe morphological defects at every age, especially in the head and midpiece. In the testicular epithelium, we saw normal numbers of cells in earlier stages of spermatogenesis, but reduced numbers of round spermatids in Cstf2t(-/-) mice. Although Leydig cell numbers were normal, we did observe reduced levels of plasma testosterone in the knockout animals, suggesting that reduced androgen might also be contributing to the Cstf2t(-/-) phenotype. Finally, while τCstF-64 was expressed in a variety of immune cell types in wild type mice, we did not find differences in secreted IgG or IgM or changes in immune cell populations in Cstf2t(-/-) mice, suggesting that τCstF-64 function in immune cells is either redundant or vestigial. Together, these data show that τCstF-64 function is primarily to support spermatogenesis, but only incidentally to support immune cell function.

Chronic exposure to a TLR ligand injures hematopoietic stem cells.

Hematopoietic stem cells (HSC) can be harmed by disease, chemotherapy, radiation, and normal aging. We show in this study that damage also occurs in mice repeatedly treated with very low doses of LPS. Overall health of the animals was good, and there were relatively minor changes in marrow hematopoietic progenitors. However, HSC were unable to maintain quiescence, and transplantation revealed them to be myeloid skewed. Moreover, HSC from treated mice were not sustained in serial transplants and produced lymphoid progenitors with low levels of the E47 transcription factor. This phenomenon was previously seen in normal aging. Screening identified mAbs that resolve HSC subsets, and relative proportions of these HSC changed with age and/or chronic LPS treatment. For example, minor CD150(Hi)CD48(-) populations lacking CD86 or CD18 expanded. Simultaneous loss of CD150(Lo/-)CD48(-) HSC and gain of the normally rare subsets, in parallel with diminished transplantation potential, would be consistent with age- or TLR-related injury. In contrast, HSC in old mice differed from those in LPS-treated animals with respect to VCAM-1 or CD41 expression and lacked proliferation abnormalities. HSC can be exposed to endogenous and pathogen-derived TLR ligands during persistent low-grade infections. This stimulation might contribute in part to HSC senescence and ultimately compromise immunity.

E47 regulates hematopoietic stem cell proliferation and energetics but not myeloid lineage restriction.

The immune system is replenished by self-renewing hematopoietic stem cells (HSCs) that produce multipotent progenitors (MPPs) with little renewal capacity. E-proteins, the widely expressed basic helix-loop-helix transcription factors, contribute to HSC and MPP activity, but their specific functions remain undefined. Using quantitative in vivo and in vitro approaches, we show that E47 is dispensable for the short-term myeloid differentiation of HSCs but regulates their long-term capabilities. E47-deficient progenitors show competent myeloid production in short-term assays in vitro and in vivo. However, long-term myeloid and lymphoid differentiation is compromised because of a progressive loss of HSC self-renewal that is associated with diminished p21 expression and hyperproliferation. The activity of E47 is shown to be cell-intrinsic. Moreover, E47-deficient HSCs and MPPs have altered expression of genes associated with cellular energy metabolism, and the size of the MPP pool but not downstream lymphoid precursors in bone marrow or thymus is rescued in vivo by antioxidant. Together, these observations suggest a role for E47 in the tight control of HSC proliferation and energy metabolism, and demonstrate that E47 is not required for short-term myeloid differentiation.

Molecular resolution of the B cell landscape.

The progression of hematopoietic stem cells (HSCs) to the B lymphocyte lineage requires that uncommitted progenitors successfully negotiate the transition from multipotency to unipotency, including the loss of self-renewal potential. Previous work identified essential transcription factors that mediate B lineage development. Major advances build on this knowledge and reveal coordinated changes in gene expression occurring within single cells at sequential stages in the B cell differentiation pathway. Recent studies on epigenetic mechanisms also provide a framework within which transcription factor activity, chromatin modifications, and gene expression patterns can be viewed at hierarchical levels to link genotype and phenotype.

Naïve and memory B cells in the rhesus macaque can be differentiated by surface expression of CD27 and have differential responses to CD40 ligation.

The rhesus macaque (RM) model has the potential to be an invaluable tool for studying B cell populations during pathogenic infections, however, to date, there has been no definitive delineation of naïve and memory B cell populations in the RM. This has precluded a rigorous analysis of the generation, persistence and resolution of a pathogen-specific memory B cell response. The present study utilized multiple analyses to demonstrate that CD27 expression on B cells is consistent with a memory phenotype. Compared to CD20+CD27- B cells, CD20+CD27+ B cells were larger in size, and preferentially accumulated at effector sites. Direct sequence analysis revealed that CD20+CD27+ B cells had an increased frequency of point mutations that were consistent with somatic hypermutation and at a functional level, CD40 ligation improved CD20+CD27- but not CD20+CD27+ B cell survival in vitro. These data provide definitive evidence that the naïve and memory B cell populations of the RM can be differentiated using surface expression of CD27.

C/EBPbeta regulates transcription factors critical for proliferation and survival of multiple myeloma cells.

CCAAT/enhancer-binding protein beta (C/EBPbeta), also known as nuclear factor-interleukin-6 (NF-IL6), is a transcription factor that plays an important role in the regulation of growth and differentiation of myeloid and lymphoid cells. Mice deficient in C/EBPbeta show impaired generation of B lymphocytes. We show that C/EBPbeta regulates transcription factors critical for proliferation and survival in multiple myeloma. Multiple myeloma cell lines and primary multiple myeloma cells strongly expressed C/EBPbeta, whereas normal B cells and plasma cells had little or no detectable levels of C/EBPbeta. Silencing of C/EBPbeta led to down-regulation of transcription factors such as IRF4, XBP1, and BLIMP1 accompanied by a strong inhibition of proliferation. Further, silencing of C/EBPbeta led to a complete down-regulation of antiapoptotic B-cell lymphoma 2 (BCL2) expression. In chromatin immunoprecipitation assays, C/EBPbeta directly bound to the promoter region of IRF4, BLIMP1, and BCL2. Our data indicate that C/EBPbeta is involved in the regulatory network of transcription factors that are critical for plasma cell differentiation and survival. Targeting C/EBPbeta may provide a novel therapeutic strategy in the treatment of multiple myeloma.