Fetal Lymphoid Progenitors Become Restricted to B-1 Fates Coincident with IL-7Rα Expression.

B-1 cells represent a sub-fraction of B lymphocytes that participate in T cell-independent antibody production and contribute to innate immunity. While the production of B-1 cells is favored during the fetal waves of lymphopoiesis, it has been unclear when and how that differentiation option is specified. To clarify this, lymphoid and hematopoietic progenitors of fetal liver (FL) and adult bone marrow (ABM) were examined for the B cell differentiation potential. Mouse common lymphoid progenitors (CLPs) and more primitive KSL fraction of FL and ABM were transferred to SCID mice and donor-derived B cell subsets were analyzed 4 weeks later. CLPs were also cultured on ST2 stromal cells for 6 days prior to transplantation. While Lin- IL-7Rα+ CLPs from ABM differentiated to B-1, B-2 and marginal zone B (MZB) cells, equivalent cells from d15 FL differentiated mostly to B-1a cells. We found that fetal CLPs had less ability to colonize the bone marrow than adult CLPs. However, the fetal/adult difference was already present when progenitors were cultured in an identical condition before transplantation. More primitive KSL fraction of FL could generate the same broad spectrum of B cells typical of adults, including splenic MZB cells. In conclusion, we argue that FL and ABM-CLPs are intrinsically different regarding B-1/B-2 fates and the difference is acquired just before or coincident with the acquisition of IL-7Rα expression.

BANK1 Regulates IgG Production in a Lupus Model by Controlling TLR7-Dependent STAT1 Activation.

The purpose of our study was to investigate the effects of the adaptor Bank1 in TLR7 signaling using the B6.Sle1.yaa mouse, a lupus model that develops disease through exacerbated TLR7 expression. Crosses of B6.Sle1.yaa with Bank1-/- mice maintained several B and myeloid cell phenotypes close to normal wild-type levels. Most striking was the reduction in total serum IgG antibodies, but not of IgM, and reduced serum levels of autoantibodies, IL-6, and BAFF. Bank1 deficiency did modify numbers of MZ B cells and total B cell numbers, as well as expression of CXCR4 by follicular helper T cells. Other T cell changes were not observed. Bank1 deficiency did not modify numbers of germinal center B cells or plasma cells or clinical disease outcomes. Purified B cells from Bank1 deficient mice had strongly reduced Ifnb, Ifna4, Irf7, Aicda and Stat1 gene expression following TLR7 agonist stimulation. Interestingly, phosphorylation of Tyr701, but not of Ser727 of STAT1, was impaired in splenic B cells from B6.Sle1.yaa.Bank1-/- mice, as was the nuclear translocation of IRF7 in response to TLR7 agonist stimulation. Further, Bank1 deficiency in B6.Sle1.yaa mice reduced the production of IgG2c after in vitro TLR7 agonist stimulation. Our results demonstrate that Bank1 controls TLR7-mediated type I interferon production. Combined with the control of the nuclear translocation of IRF7, the modulation of STAT1 transcription and phosphorylation, Bank1 contributes to IgG production during development of autoimmune disease.

Stem and progenitor cell subsets are affected by JAK2 signaling and can be monitored by flow cytometry.

Although extremely rare, hematopoietic stem cells (HSCs) are divisible into subsets that differ with respect to differentiation potential and cell surface marker expression. For example, we recently found that CD86(-) CD150(+) CD48(-) HSCs have limited potential for lymphocyte production. This could be an important new tool for studying hematological abnormalities. Here, we analyzed HSC subsets with a series of stem cell markers in JAK2V617F transgenic (Tg) mice, where the mutation is sufficient to cause myeloproliferative neoplasia with lymphocyte deficiency. Total numbers of HSC were elevated 3 to 20 fold in bone marrow of JAK2V617F mice. Careful analysis suggested the accumulation involved multiple HSC subsets, but particularly those characterized as CD150(HI) CD86(-) CD18(L)°CD41(+) and excluding Hoechst dye. Real-Time PCR analysis of their HSC revealed that the erythropoiesis associated gene transcripts Gata1, Klf1 and Epor were particularly high. Flow cytometry analyses based on two differentiation schemes for multipotent progenitors (MPP) also suggested alteration by JAK2 signals. The low CD86 on HSC and multipotent progenitors paralleled the large reductions we found in lymphoid progenitors, but the few that were produced functioned normally when sorted and placed in culture. Either of two HSC subsets conferred disease when transplanted. Thus, flow cytometry can be used to observe the influence of abnormal JAK2 signaling on stem and progenitor subsets. Markers that similarly distinguish categories of human HSCs might be very valuable for monitoring such conditions. They could also serve as indicators of HSC fitness and suitability for transplantation.

RAG-1 and Ly6D independently reflect progression in the B lymphoid lineage.

Common lymphoid progenitors (CLPs) are thought to represent major intermediates in the transition of hematopoietic stem cells (HSCs) to B lineage lymphocytes. However, it has been obvious for some time that CLPs are heterogeneous, and there has been controversy concerning their differentiation potential. We have now resolved four Flt3(+) CLP subsets that are relatively homogenous and capable of forming B cells. Differentiation potential and gene expression patterns suggest Flt3(+) CLPs lacking both Ly6D and RAG-1 are the least differentiated. In addition to B cells, they generate natural killer (NK) and dendritic cells (DCs). At the other extreme is a subset of the recently described Flt3(+) Ly6D(+) CLPs that have a history of RAG-1 expression and are B lineage restricted. These relatively abundant and potent CLPs were depleted within 48 hours of acute in vivo estrogen elevation, suggesting they descend from hormone regulated progenitors. This contrasts with the hormone insensitivity of other CLP subsets that include NK lineage progenitors. This progenitor heterogeneity and differentiation complexity may add flexibility in response to environmental changes. Expression of RAG-1 and display of Ly6D are both milestone events, but they are neither synchronized nor dependent on each other.

The Satb1 protein directs hematopoietic stem cell differentiation toward lymphoid lineages.

How hematopoietic stem cells (HSCs) produce particular lineages is insufficiently understood. We searched for key factors that direct HSC to lymphopoiesis. Comparing gene expression profiles for HSCs and early lymphoid progenitors revealed that Satb1, a global chromatin regulator, was markedly induced with lymphoid lineage specification. HSCs from Satb1-deficient mice were defective in lymphopoietic activity in culture and failed to reconstitute T lymphopoiesis in wild-type recipients. Furthermore, Satb1 transduction of HSCs and embryonic stem cells robustly promoted their differentiation toward lymphocytes. Whereas genes that encode Ikaros, E2A, and Notch1 were unaffected, many genes involved in lineage decisions were regulated by Satb1. Satb1 expression was reduced in aged HSCs with compromised lymphopoietic potential, but forced Satb1 expression partly restored that potential. Thus, Satb1 governs the initiating process central to the replenishing of lymphoid lineages. Such activity in lymphoid cell generation may be of clinical importance and useful to overcome immunosenescence.

Early events in lymphopoiesis: an update.

PURPOSE OF REVIEW: Cells of the immune system are replaced in large numbers throughout life, and the underlying mechanisms have been extensively studied. Whereas the pace of discovery in this area is unprecedented, many questions remain, particularly with respect to lymphocyte formation. RECENT FINDINGS: While transcription factors have long been a focus of investigation, microRNAs are also being implicated in lymphopoiesis. Lymphocytes are normally replaced in correct proportion to other blood cells, but ratios change dramatically during infections. Long-standing issues relating to T versus B lineage divergence remain but have been enriched with remarkable new findings about thymus seeding. There are indications that at least some age-related changes in lymphopoiesis may be reversible. Finally, knowledge obtained from studies of mice is slowly being extended to humans. SUMMARY: We can now appreciate that new lymphoid progenitors are drawn from a heterogeneous collection of hematopoietic stem cells through asynchronous patterns of gene expression. Complex interactions then occur between the gene products, preparing lymphoid progenitors to respond to environmental cues. Whereas unique markers describe the process of lymphocyte formation in humans, fundamental information now available should suggest ways to promote rebound from chemotherapy or transplantation and reverse declines associated with aging.

Nuclear transferred embryonic stem cells for analysis of B1 B-lymphocyte development.

The transfer of nuclei of fully differentiated cells into enucleated oocytes is a well-recognized method for the generation of embryonic stem (ES) cells. Here, we demonstrate that nuclear transferred ES (NT-ES) cells can be established with high efficiency using innate-like B lymphocytes as donor cells. We established two mouse lines carrying rearranged immunoglobulin heavy and light chains using NT-ES cells containing nuclei from peritoneal cavity B1 cells. Analysis of B1 clone lines revealed that the B1-cell generation critically depends on the interaction between antigen (possibly self-antigen) and surface immunoglobulin, while the B1-cell maintenance requires the peritoneal environment. The B1-cell expansion takes place in spleen, and is held in check by competitor B2 cells. The results indicate that the NT-ES method could replace the transgenic or knock-in mouse approaches currently used to study the biology of cells that undergo somatic rearrangements of their antigen receptor genes.

Bone marrow endothelial progenitors augment atherosclerotic plaque regression in a mouse model of plasma lipid lowering.

The major event initiating atherosclerosis is hypercholesterolemia-induced disruption of vascular endothelium integrity. In settings of endothelial damage, endothelial progenitor cells (EPCs) are mobilized from bone marrow into circulation and home to sites of vascular injury where they aid endothelial regeneration. Given the beneficial effects of EPCs in vascular repair, we hypothesized that these cells play a pivotal role in atherosclerosis regression. We tested our hypothesis in the atherosclerosis-prone mouse model in which hypercholesterolemia, one of the main factors affecting EPC homeostasis, is reversible (Reversa mice). In these mice, normalization of plasma lipids decreased atherosclerotic burden; however, plaque regression was incomplete. To explore whether endothelial progenitors contribute to atherosclerosis regression, bone marrow EPCs from a transgenic strain expressing green fluorescent protein (GFP) under the control of endothelial cell-specific Tie2 promoter (Tie2-GFP(+)) were isolated. These cells were then adoptively transferred into atheroregressing Reversa recipients where they augmented plaque regression induced by reversal of hypercholesterolemia. Advanced plaque regression correlated with engraftment of Tie2-GFP(+) EPCs into endothelium and resulted in an increase in atheroprotective nitric oxide and improved vascular relaxation. Similarly augmented plaque regression was also detected in regressing Reversa mice treated with the stem cell mobilizer AMD3100 which also mobilizes EPCs to peripheral blood. We conclude that correction of hypercholesterolemia in Reversa mice leads to partial plaque regression that can be augmented by AMD3100 treatment or by adoptive transfer of EPCs. This suggests that direct cell therapy or indirect progenitor cell mobilization therapy may be used in combination with statins to treat atherosclerosis.

CD86 is expressed on murine hematopoietic stem cells and denotes lymphopoietic potential.

A unique subset of CD86(-) HSCs was previously discovered in mice that were old or chronically stimulated with lipopolysaccharide. Functionally defective HSCs were also present in those animals, and we now show that CD86(-) CD150(+) CD48(-) HSCs from normal adult mice are particularly poor at restoring the adaptive immune system. Levels of the marker are high on all progenitors with lymphopoietic potential, and progressive loss helps to establish relations between progenitors corresponding to myeloid and erythroid lineages. CD86 represents an important tool for subdividing HSCs in several circumstances, identifying those unlikely to generate a full spectrum of hematopoietic cells.

Replenishing B lymphocytes in health and disease.

The path from hematopoietic stem cells (HSCs) to functional B lymphocytes has long been appreciated as a basic model of differentiation, but much clinically relevant information has also been obtained. It is now possible to conduct single cell studies with increasingly high resolution, revealing that individual stem and progenitor cells differ from each other with respect to differentiation potential and fates. B lymphopoiesis is now seen as a gradual and unsynchronized process where progenitors eventually become B lineage restricted. Major milestones have been identified, but a precise sequence need not be followed and oscillation between states is possible. It is not yet clear if this versatility has survival value, but information is accumulating about infections and age-related changes.

In vivo expansion of CD4+Foxp3+ regulatory T cells mediated by GITR molecules.

CD4(+)Foxp3(+) regulatory T cells (Treg cells) play an important role in maintaining self-tolerance as suppressive/regulatory CD4 T cells. In vitro analyses have revealed the characteristics of Treg cells, that is, hyporesponsiveness when stimulated via TCR in the presence of splenic APC. In this study, we report a new mAb, G3c, which can induce the expansion of Treg cells stimulated with anti-CD3 Ab along with splenic APC, the culture conditions in which Treg cells exhibit hyporesponsiveness. Surprisingly, G3c mAb recognized glucocorticoid-induced TNFR family-related proteins (GITR). G3c mAb had stronger co-stimulatory activity for both Treg cells and responder T cells than another anti-GITR Ab (DTA1) in vitro. The in vivo administration of G3c increased the number of Treg cells and had less effect in inducing anti-tumor immunity in normal mice, although G3c had some anti-tumor effect on non-Treg cells in the absence of Treg cells in vivo, in contrast to the anti-tumor therapeutic effect of DTA1 in normal mice. Therefore, we need to know that the manipulation of immune responses with the use of anti-GITR Abs results from a balance between co-stimulatory effects on Treg cells and on responder cells, and we must aim at a narrow window leading to the therapeutic effects.

Antigen-independent generation of a unique CD4 T cell-subset with aging and its persistent unresponsiveness.

Aging leads to a decline in the reactivity of CD4 T cells, in humans and mice. However, we have reported that not all CD4 T cells in aged mice were hyporesponsive, that is, a particular subset maintained the ability to mount a normal response. In this study, we examined the possibility of recalling reactivity in the hyporesponsive CD4 T cell-subset in aged mice. In vivo experiments revealed the changes in CD4 T cell-subsets in aged mice to be antigen-independent and aging-dependent. Once the CD4 T cells became hyporesponsive, they persistently exhibited a weak response. Furthermore, immunization with a co-stimulatory antibody had no effect on T cell-responses in aged mice, although it had a significant effect in young mice. As this hyporesponsive subset accounts for the majority of CD4 T cells in aged mice, it is important to elucidate the cause of the hyporesponsiveness.

CD4+CD25+Foxp3+ T cells and CD4+CD25-Foxp3+ T cells in aged mice.

Aging is associated with a progressive decline in T cell-mediated immune responses. However, it has been unknown whether regulatory/suppressive CD4 T cells are involved in this decline. Our in vitro analyses revealed that CD4+CD25+ T cells, the well-characterized naturally occurring regulatory/suppressive CD4 T cells, in aged mice are functionally comparable to those in young mice (i.e., anergic and suppressive), although slightly increased in number. In contrast, functional changes to whole CD4+CD25- T cells were pronounced in aged mice, i.e., the majority of aged CD4+CD25- T cells exhibited a significant hyporesponsiveness, and the remaining cells maintained a normal responsiveness. Furthermore, we identified Foxp3 (a transcription factor critical in conferring the regulatory/suppressive function to CD4 T cells)-positive suppressive CD4 T cells among aged hyporesponsive CD4+CD25- T cells. These results suggest that the age-related decline in T cell-mediated immune responses is ascribable to changes in the CD4+CD25- T cell population and not to a functional augmentation of suppressive CD4+CD25+ T cells.

Cross-linking of CD45 on suppressive/regulatory T cells leads to the abrogation of their suppressive activity in vitro.

CD4(+)CD25(+) T cells have immunoregulatory and suppressive functions and are responsible for suppressing self-reactive cells and maintaining self-tolerance. In addition to CD4(+)CD25(+) T cells, there is some evidence that a fraction of CD4(+)CD25(-) T cells exhibit suppressive activity in vitro or in vivo. We have shown, using aged mice, that aging not only leads to a decline in the ability to mount CD4(+)CD25(-) T cell responses, but, at the same time, renders aged CD4(+)CD25(-) T cells suppressive. In this study we report two newly established mAbs that could abrogate the suppressive function of aged CD4(+)CD25(-) T cells. These mAbs recognized the same protein, the transmembrane phosphatase CD45. Cross-linking of CD45 on aged CD4(+)CD25(-) T cells was required for the disruption of their suppressive activity. Surprisingly, these mAbs also abrogated the suppressive action of CD4(+)CD25(+) T cells in vitro. Our results demonstrate an unexpected function of CD45 as a negative regulator neutralizing the suppressive activity of aged CD4(+)CD25(-) and young CD4(+)CD25(+) T cells.

Expression profiling with arrays of randomly disrupted genes in mouse embryonic stem cells leads to in vivo functional analysis.

DNA arrays are capable of profiling the expression patterns of many genes in a single experiment. After finding a gene of interest in a DNA array, however, labor-intensive gene-targeting experiments sometimes must be performed for the in vivo analysis of the gene function. With random gene trapping, on the other hand, it is relatively easy to disrupt and retrieve hundreds of genes/gene candidates in mouse embryonic stem (ES) cells, but one could overlook potentially important gene-disruption events if only the nucleotide sequences and not the expression patterns of the trapped DNA segments are analyzed. To combine the benefits of the above two experimental systems, we first created approximately 900 genetrapped mouse ES cell clones and then constructed arrays of cDNAs derived from the disrupted genes. By using these arrays, we identified a novel gene predominantly expressed in the mouse brain, and the corresponding ES cell clone was used to produced mice homozygous for the disrupted allele of the gene. Detailed analysis of the knockout mice revealed that the gene trap vector completely abolished gene expression downstream of its integration site. Therefore, identification of a gene or novel gene candidate with an interesting expression pattern by using this type of DNA array immediately allows the production of knockout mice from an ES cell clone with a disrupted allele of the sequence of interest.