Plasma Cells Are Obligate Effectors of Enhanced Myelopoiesis in Aging Bone Marrow.

Aging results in increased myelopoiesis, which is linked to the increased incidence of myeloid leukemias and production of myeloid-derived suppressor cells. Here, we examined the contribution of plasma cells (PCs) to age-related increases in myelopoiesis, as PCs exhibit immune regulatory function and sequester in bone marrow (BM). PC number was increased in old BM, and they exhibited high expression of genes encoding inflammatory cytokines and pathogen sensors. Antibody-mediated depletion of PCs from old mice reduced the number of myeloid-biased hematopoietic stem cells and mature myeloid cells to levels in young animals, but lymphopoiesis was not rejuvenated, indicating that redundant mechanisms inhibit that process. PCs also regulated the production of inflammatory factors from BM stromal cells, and disruption of the PC-stromal cell circuitry with inhibitors of the cytokines IL-1 and TNF-α attenuated myelopoiesis in old mice. Thus, the age-related increase in myelopoiesis is driven by an inflammatory network orchestrated by PCs.

The layered development of mouse B and T Cells.

Traditional models of lymphopoiesis present B and T cell development as a linear process that initiates in the fetus and continues after birth in the bone marrow and thymus, respectively. However, this view of lymphocyte development is not in accord with reports, dating back several decades, indicating that the types of lymphocytes generated before and after birth differ. In this regard, selected γδ T cells, and those that utilize the Vγ3 receptor in particular, and innate-like B-1 B cells preferentially arise during fetal blood cell development. This review synthesizes data from multiple laboratories, with an emphasis on our own work using mouse models, demonstrating that innate and conventional B and T cells emerge in hematopoietic stem cell independent and dependent waves of development that are differentially regulated. This layering of lymphocyte development has implications for understanding the composition of the adult immune system and may provide insights into the origin of various lymphocytic leukemias.

Distinct Genetic Networks Orchestrate the Emergence of Specific Waves of Fetal and Adult B-1 and B-2 Development.

B cell development is often depicted as a linear process initiating in the fetus and continuing postnatally. Using a PU.1 hypomorphic mouse model, we found that B-1 and B-2 lymphopoiesis occurred in distinct fetal and adult waves differentially dependent on the Sfpi1 14 kB upstream regulatory element. The initial wave of fetal B-1 development was absent in PU.1 hypomorphic mice, while subsequent fetal and adult waves emerged. In contrast, B-2 lymphopoiesis occurred in distinct fetal and adult waves. Whole-transcriptome profiling of fetal and adult B cell progenitors supported the existence of three waves of B-1 and two waves of B-2 development and revealed that the network of transcription factors governing B lineage specification and commitment was highly divergent between B-1 and B-2 progenitors. These findings support the view that the B-1 and B-2 lineages are distinct and provide a genetic basis for layering of immune system development.

Envelope protein-specific B cell receptors direct lentiviral vector tropism in vivo.

While studying transgene expression after systemic administration of lentiviral vectors, we found that splenic B cells are robustly transduced, regardless of the types of pseudotyped envelope proteins. However, the administration of two different pseudotypes resulted in transduction of two distinct B cell populations, suggesting that each pseudotype uses unique and specific receptors for its attachment and entry into splenic B cells. Single-cell RNA sequencing analysis of the transduced cells demonstrated that different pseudotypes transduce distinct B cell subpopulations characterized by specific B cell receptor (BCR) genotypes. Functional analysis of the BCRs of the transduced cells demonstrated that BCRs specific to the pseudotyping envelope proteins mediate viral entry, enabling the vectors to selectively transduce the B cell populations that are capable of producing antibodies specific to their envelope proteins. Lentiviral vector entry via the BCR activated the transduced B cells and induced proliferation and differentiation into mature effectors, such as memory B and plasma cells. BCR-mediated viral entry into clonally specific B cell subpopulations raises new concepts for understanding the biodistribution of transgene expression after systemic administration of lentiviral vectors and offers new opportunities for BCR-targeted gene delivery by pseudotyped lentiviral vectors.

B-1 B cell development in the fetus and adult.

Models of hematopoiesis often depict lymphocyte production as a uniform process in which a homogenous population of hematopoietic stem cells (HSCs) generates progenitors from which all types of lymphocytes are derived. However, it is increasingly evident that these schemes are too simplistic and that the lymphoid potential of HSCs and precursors arising in the embryo, fetus, neonate, and adult is remarkably distinct. We review recent findings regarding the development of B lymphocytes, and the B-1 B cell lineage in particular, as a case in point. These studies show that B-1 and B-2 B cells involved in innate and adaptive immune responses, respectively, arise in staggered waves of development from distinct progenitors. We discuss the implications of this layered model of B cell development for understanding normal and dysregulated B lymphopoiesis.

Differential Expression of PU.1 and Key T Lineage Transcription Factors Distinguishes Fetal and Adult T Cell Development.

The PU.1 transcription factor plays a critical role in the regulation of T cell development, so a report that it is dispensable for fetal thymopoiesis is puzzling. To understand this paradox, we examined the requirement for PU.1, encoded by Spi1, during fetal, neonatal, and adult thymopoiesis in a PU.1 hypomorphic mouse generated by deletion of the Spi1 14-kb upstream regulatory element and by analysis of patterns of gene expression in fetal and adult T cell progenitors. Our data demonstrate that the initiation of thymopoiesis during early gestation is less dependent on PU.1 compared with T cell differentiation in adults and that fetal T cell progenitors express lower levels of Spi1 compared with their adult counterparts. We also show that expression of the core network of T lineage transcription factors regulated by PU.1 differs in fetal and adult T cell progenitors. In particular, PU.1-regulated genes that promote T cell differentiation are differentially expressed in fetal versus adult early T lineage progenitors. These results indicate that the transcriptional differences between the fetal and adult T cell developmental programs are driven in part by differential levels of PU.1 expression and that this likely underlies the differences in the properties of fetal and adult T cell progenitors.

Fetal B-cell lymphopoiesis and the emergence of B-1-cell potential.

Most B cells in peripheral lymphoid tissues are produced in adult bone marrow and are referred to as B-2 cells. A minor B-cell population, known as the B-1-cell population, that is mainly involved in innate immune responses has been identified in mice. In contrast to B-2 cells, B-1-cell progenitors are produced most efficiently during fetal life. This Review focuses on the emergence of B-1-cell potential during embryogenesis, summarizes recent advances in the delineation of a fetal B-1-cell-specified progenitor, and discusses the possibility that distinct fetal and adult B-cell developmental programmes might be operative in humans.

Shared signaling systems in myeloid cell-mediated muscle regeneration.

Much of the focus in muscle regeneration has been placed on the identification and delivery of stem cells to promote regenerative capacity. As those efforts have advanced, we have learned that complex features of the microenvironment in which regeneration occurs can determine success or failure. The immune system is an important contributor to that complexity and can determine the extent to which muscle regeneration succeeds. Immune cells of the myeloid lineage play major regulatory roles in tissue regeneration through two general, inductive mechanisms: instructive mechanisms that act directly on muscle cells; and permissive mechanisms that act indirectly to influence regeneration by modulating angiogenesis and fibrosis. In this article, recent discoveries that identify inductive actions of specific populations of myeloid cells on muscle regeneration are presented, with an emphasis on how processes in muscle and myeloid cells are co-regulated.

Murine B-1 B cell progenitors initiate B-acute lymphoblastic leukemia with features of high-risk disease.

B-1 and B-2 B cells derive from distinct progenitors that emerge in overlapping waves of development. The number of murine B-1 progenitors peaks during fetal development whereas B-2 B cell production predominates in adult bone marrow. Many genetic mutations that underlie B-acute lymphoblastic leukemia (B-ALL) occur in the fetus, at which time B-1 progenitor numbers are high. However, whether B-ALL can initiate in B-1 progenitors is unknown. In the present study, we report that BCR-ABL-transformed murine B-1 progenitors can be B-ALL cells of origin and demonstrate that they initiate disease more rapidly than do oncogene-expressing B-2 progenitors. We further demonstrate that B-1 progenitors exhibit relative resistance to apoptosis and undergo significant growth following oncogene expression, and we propose that these properties underlie the accelerated kinetics with which they initiate leukemia. These results provide a developmental perspective on the origin of B-ALL and indicate B cell lineage as a factor influencing disease progression.

Genetic regulation of thymocyte progenitor aging.

The number of T cell progenitors is significantly reduced in the involuted thymus, and the growth and developmental potential of the few cells that are present is severely attenuated. This review provides an overview of how aging affects T cell precursors before and following entry into the thymus and discusses the age-related genetic changes that may occur in them. Finally, interventions that rejuvenate thymopoiesis in the elderly by targeting T cell progenitors are discussed.

Fibroblast growth factor-7 partially reverses murine thymocyte progenitor aging by repression of Ink4a.

Involution of the thymus results in reduced production of naive T cells, and this in turn is thought to contribute to impaired immunity in the elderly. Early T-cell progenitors (ETPs), the most immature intrathymic T-cell precursors, harvested from the involuted thymus exhibit a diminished proliferative potential and increased rate of apoptosis and as a result their number is significantly reduced. In the present study, we show that these age-induced alterations result in part from increased expression of the Ink4a tumor-suppressor gene in ETPs. We also show that repression of Ink4a in aged ETPs results in their partial rejuvenation and that this can be accomplished by in vivo fibroblast growth factor 7 administration. These results define a genetic basis for thymocyte progenitor aging and demonstrate that the senescence-associated gene Ink4a can be pharmacologically repressed in ETPs to partially reverse the effects of aging.

Reduced production of B-1-specified common lymphoid progenitors results in diminished potential of adult marrow to generate B-1 cells.

B-1 B cells have been proposed to be preferentially generated from fetal progenitors, but this view is challenged by studies concluding that B-1 production is sustained throughout adult life. To address this controversy, we compared the efficiency with which hematopoietic stem cells (HSCs) and common lymphoid progenitors (CLPs) from neonates and adults generated B-1 cells in vivo and developed a clonal in vitro assay to quantify B-1 progenitor production from CLPs. Adult HSCs and CLPs generated fewer B-1 cells in vivo compared with their neonatal counterparts, a finding corroborated by the clonal studies that showed that the CLP compartment includes B-1- and B-2-specified subpopulations and that the former cells decrease in number after birth. Together, these data indicate that B-1 lymphopoiesis is not sustained at constant levels throughout life and define a heretofore unappreciated developmental heterogeneity within the CLP compartment.

Embryonic day 9 yolk sac and intra-embryonic hemogenic endothelium independently generate a B-1 and marginal zone progenitor lacking B-2 potential.

The majority of B lymphocytes in the adult mouse are generated in the bone marrow from hematopoietic stem cells (HSCs) that first appear in the aorta-gonado-mesonephros region of the fetus on embryonic day (E) 10.5-11. Comparatively less is known about B-cell development during embryogenesis. For example, which specific embryonic tissues participate in B lymphopoiesis and whether hematopoietic differentiation is skewed toward specific B-cell subsets in the embryo are unanswered questions, because the systemic circulation is initiated early during embryogenesis, resulting in an admixture of cells potentially originating from multiple sites. We demonstrate, using Ncx1(-/-) mice that lack systemic blood circulation, that the E9 yolk sac (YS) and the intra-embryonic para-aortic splanchnopleura (P-Sp) tissues independently give rise to AA4.1(+)CD19(+)B220(lo-neg) B progenitor cells that preferentially differentiate into innate type B-1 and marginal zone (MZ) B cells but not into B-2 cells upon transplantation. We have further demonstrated that these B-1 progenitor cells arise directly from YS and P-Sp hemogenic endothelium. These results document the initial wave of innate B lymphopoietic progenitor cells available for seeding the fetal liver at E11. The results of these studies expand our knowledge of hemogenic endothelial sites specifying distinct B-1 and MZ cell fates apart from B-2 cells and independent of an HSC origin during development.

Transient PU.1 low fetal progenitors generate lymphoid progeny that contribute to adult immunity.

Hematopoietic stem cells and multipotential progenitors emerge in multiple, overlapping waves of fetal development. Some of these populations seed the bone marrow and sustain adult B- and T-cell development long-term after birth. However, others are present transiently, but whether they are vestigial or generate B and T cells that contribute to the adult immune system is not well understood. We now report that transient fetal progenitors distinguished by expression of low levels of the PU.1 transcription factor generated activated and memory T and B cells that colonized and were maintained in secondary lymphoid tissues. These included the small and large intestines, where they may contribute to the maintenance of gut homeostasis through at least middle age. At least some of the activated/memory cells may have been the progeny of B-1 and marginal zone B cells, as transient PU.1low fetal progenitors efficiently generated those populations. Taken together, our data demonstrate the potential of B- and T-cell progeny of transient PU.1low fetal progenitors to make an early and long-term contribution to the adult immune system.

Formation of B-1 B cells from neonatal B-1 transitional cells exhibits NF-κB redundancy.

The stages of development leading up to the formation of mature B-1 cells have not been identified. As a result, there is no basis for understanding why various genetic defects, and those in the classical or alternative NF-κB pathways in particular, differentially affect the B-1 and B-2 B cell lineages. In this article, we demonstrate that B-1 B cells are generated from transitional cell intermediates that emerge in a distinct neonatal wave of development that is sustained for ~2 wk after birth and then declines as B-2 transitional cells predominate. We further show that, in contrast to the dependence of B-2 transitional cells on the alternative pathway, the survival of neonatal B-1 transitional cells and their maturation into B-1 B cells occurs as long as either alternative or classical NF-κB signaling is intact. On the basis of these results, we have generated a model of B-1 development that allows the defects in B-1 and B-2 cell production observed in various NF-κB-deficient strains of mice to be placed into a coherent cellular context.

Immature B-cell progenitors survive oncogenic stress and efficiently initiate Ph+ B-acute lymphoblastic leukemia.

Philadelphia chromosome-positive (Ph(+)) B-acute lymphoblastic leukemia (B-ALL) can initiate in committed B-cell progenitors. However, the stages of B-cell differentiation in which disease can initiate and the efficiency with which this occurs are unclear. We now demonstrate that B-cell progenitors, up to and including the pro-B cell, efficiently initiate Ph(+) B-ALL. However, cells at the pre-B-cell stage of development did not initiate disease. We show that this difference in leukemia initiating potential is due to the level at which the Arf tumor suppressor gene is induced in specific stages of B lymphopoiesis. Whereas immature B-cell progenitors survive the relatively low levels of Arf that are induced after oncogene expression, pre-B cells express the tumor suppressor gene at high levels and undergo massive apoptosis. These data demonstrate that the molecular events that control Ph(+) B-ALL initiation and tumor suppression in the B-cell lineage are developmentally regulated.

Myeloid cell-specific mutation of Spi1 selectively reduces M2-biased macrophage numbers in skeletal muscle, reduces age-related muscle fibrosis and prevents sarcopenia.

Intramuscular macrophages play key regulatory roles in determining the response of skeletal muscle to injury and disease. Recent investigations showed that the numbers and phenotype of intramuscular macrophages change during aging, suggesting that those changes could influence the aging process. We tested that hypothesis by generating a mouse model that harbors a myeloid cell-specific mutation of Spi1, which is a transcription factor that is essential for myeloid cell development. The mutation reduced the numbers of macrophages biased to the CD163+/CD206+ M2 phenotype in muscles of aging mice without affecting the numbers of CD68-expressing macrophages and reduced the expression of transcripts associated with the M2-biased phenotype. The mutation did not affect the colony-forming ability or the frequency of specific subpopulations of bone marrow hematopoietic cells and did not affect myeloid/lymphoid cell ratios in peripheral blood leukocyte populations. Cellularity of most myeloid lineage cells was not influenced by the mutation. The Spi1 mutation in bone marrow-derived macrophages in vitro also did not affect expression of transcripts that indicate the M2-biased phenotype. Thus, myeloid cell-targeted mutation of Spi1 influences macrophage phenotype in muscle but did not affect earlier stages of differentiation of cells in the macrophage lineage. The mutation reduced age-related muscle fibrosis, which is consistent with the reduction of M2-biased macrophages, and reduced expression of the pro-fibrotic enzyme arginase. Most importantly, the mutation prevented sarcopenia. Together, our observations indicate that intramuscular, M2-biased macrophages play significant roles in promoting detrimental, age-related changes in muscle.

Use of Busulfan to Condition Mice for Bone Marrow Transplantation.

Myeloablative gamma irradiation has traditionally been used to condition mice prior to bone marrow transplantation. However, irradiation induces high levels of inflammation that may alter patterns of reconstitution. In addition, gamma irradiators are being removed from many facilities for security reasons. Alternative conditioning regimens are thus needed. Here, we describe a protocol for the use of busulfan to condition mice for bone marrow transplantation and several of the variables to consider for effective implementation. For complete details on the use and execution of this protocol, please refer to Montecino-Rodriguez et al. (2019).