Maho Nagasawa

The ETS Transcription Factor Spi-B Is Required for Human Plasmacytoid Dendritic Cell Development

A number of transcription factors that act as molecular switches for hematopoietic lineage decisions have been identified. We recently described the ETS transcription factor Spi-B to be exclusively expressed in plasmacytoid dendritic cells (pDCs), but not in myeloid DCs. To assess whether Spi-B is required for pDC development we used an RNA interference knock down approach to specifically silence Spi-B protein synthesis in CD34+ precursor cells. We observed that a knock down of Spi-B mRNA strongly inhibited the ability of CD34+ precursor cells to develop into pDCs in both in vitro assays as well as in vivo upon injection into recombination activating gene 2−/− γ common−/− mice. The observed effects were restricted to the pDC lineage as the differentiation of pro–B cells and CD14+ myeloid cells was not inhibited but slightly elevated by Spi-B knock down. Knock down of the related ETS factor PU.1 also inhibited in vitro development of CD34+ cells into pDCs. However, in contrast to Spi-B, PU.1 knock down inhibited B cell and myeloid cell development as well. These results identify Spi-B as a key regulator of human pDC development.

STAT3-Mediated Up-Regulation of BLIMP1 Is Coordinated with BCL6 Down-Regulation to Control Human Plasma Cell Differentiation

STAT family members have been implicated in regulating the balance between B cell lymphoma (BCL)6 and B lymphocyte induced maturation protein (BLIMP)1 to control plasma cell differentiation. We previously showed that STAT5 induces BCL6 to block plasma cell differentiation and extend the life span of human B cells. The heterogeneity in STAT activation by cytokines and their effects on B cell differentiation prompted us to investigate the effect of STAT3 activation in plasma cell differentiation. First stimulation with IL-21, which promotes plasma cell differentiation, induced robust and prolonged STAT3 activation in primary human B cells. We then investigated effects of direct STAT3 activation on regulation of plasma cell genes, cellular phenotype, and Ig production. Activation of a tamoxifen-regulated STAT3-estrogen receptor fusion protein triggered BLIMP1 mRNA and protein up-regulation, plasma cell phenotypic features, and Ig secretion. When STAT3 was activated by IL-21 in B cells ectopically expressing BCL6, BLIMP1 was up-regulated, but only partial plasma cell differentiation was achieved. Lastly, through coexpression of BCL6 and STAT3-ER, we verified that STAT3 activation functionally mimicked IL-21 treatment and that STAT3-mediated BLIMP1 up-regulation occurred despite high BCL6 expression levels indicating that BCL6 is not the dominant repressor of BLIMP1. Thus, up-regulation of BLIMP1 alone is not sufficient for differentiation of primary human B cells into plasma cells; concomitant down-regulation of BCL6 is absolutely required for completion of the plasma cell differentiation program.

IL-1β, IL-4 and IL-12 control the fate of group 2 innate lymphoid cells in human airway inflammation in the lungs

Group 2 innate lymphoid cells (ILC2s) secrete type 2 cytokines, which protect against parasites but can also contribute to a variety of inflammatory airway diseases. We report here that interleukin 1β (IL-1β) directly activated human ILC2s and that IL-12 induced the conversion of these activated ILC2s into interferon-γ (IFN-γ)-producing ILC1s, which was reversed by IL-4. The plasticity of ILCs was manifested in diseased tissues of patients with severe chronic obstructive pulmonary disease (COPD) or chronic rhinosinusitis with nasal polyps (CRSwNP), which displayed IL-12 or IL-4 signatures and the accumulation of ILC1s or ILC2s, respectively. Eosinophils were a major cellular source of IL-4, which revealed cross-talk between IL-5-producing ILC2s and IL-4-producing eosinophils. We propose that IL-12 and IL-4 govern ILC2 functional identity and that their imbalance results in the perpetuation of type 1 or type 2 inflammation.

Functional CD47/signal regulatory protein alpha (SIRPα) interaction is required for optimal human T- and natural killer- (NK) cell homeostasis in vivo

The homeostatic control mechanisms regulating human leukocyte numbers are poorly understood. Here, we assessed the role of phagocytes in this process using human immune system (HIS) BALB/c Rag2−/−IL-2Rγc−/− mice in which human leukocytes are generated from transplanted hematopoietic progenitor cells. Interactions between signal regulatory protein alpha (SIRPα; expressed on phagocytes) and CD47 (expressed on hematopoietic cells) negatively regulate phagocyte activity of macrophages and other phagocytic cells. We previously showed that B cells develop and survive robustly in HIS mice, whereas T and natural killer (NK) cells survive poorly. Because human CD47 does not interact with BALB/c mouse SIRPα, we introduced functional CD47/SIRPα interactions in HIS mice by transducing mouse CD47 into human progenitor cells. Here, we show that this procedure resulted in a dramatic and selective improvement of progenitor cell engraftment and human T- and NK-cell homeostasis in HIS mouse peripheral lymphoid organs. The amount of engrafted human B cells also increased but much less than that of T and NK cells, and total plasma IgM and IgG concentrations increased 68- and 35-fold, respectively. Whereas T cells exhibit an activated/memory phenotype in the absence of functional CD47/SIRPα interactions, human T cells accumulated as CD4+ or CD8+ single-positive, naive, resting T cells in the presence of functional CD47/SIRPα interactions. Thus, in addition to signals mediated by T cell receptor (TCR)/MHC and/or IL/IL receptor interactions, sensing of cell surface CD47 expression by phagocyte SIRPα is a critical determinant of T- and NK-cell homeostasis under steady-state conditions in vivo.

Development of human plasmacytoid dendritic cells depends on the combined action of the basic helix‐loop‐helix factor E2‐2 and the Ets factor Spi‐B

Plasmacytoid dendritic cells (pDC) are central players in the innate and adaptive immune response against viral infections. The molecular mechanism that underlies pDC development from progenitor cells is only beginning to be elucidated. Previously, we reported that the Ets factor Spi‐B and the inhibitors of DNA binding protein 2 (Id2) or Id3, which antagonize E‐protein activity, are crucially involved in promoting or impairing pDC development, respectively. Here we show that the basic helix‐loop‐helix protein E2‐2 is predominantly expressed in pDC, but not in their progenitor cells or conventional DC. Forced expression of E2‐2 in progenitor cells stimulated pDC development. Conversely, inhibition of E2‐2 expression by RNA interference impaired the generation of pDC suggesting a key role of E2‐2 in development of these cells. Notably, Spi‐B was unable to overcome the Id2 enforced block in pDC development and moreover Spi‐B transduced pDC expressed reduced Id2 levels. This might indicate that Spi‐B contributes to pDC development by promoting E2‐2 activity. Consistent with notion, simultaneous overexpression of E2‐2 and Spi‐B in progenitor cells further stimulated pDC development. Together our results provide additional insight into the transcriptional network controlling pDC development as evidenced by the joint venture of E2‐2 and Spi‐B.

T cell–independent development and induction of somatic hypermutation in human IgM+IgD+CD27+ B cells

IgM+IgD+CD27+ B cells from peripheral blood have been described as circulating marginal zone B cells. It is still unknown when and where these cells develop. These IgM+IgD+CD27+ B cells exhibit somatic hypermutations (SHMs) in their B cell receptors, but the exact nature of the signals leading to induction of these SHMs remains elusive. Here, we show that IgM+IgD+CD27+ B cells carrying SHMs are observed during human fetal development. To examine the role of T cells in human IgM+IgD+CD27+ B cell development we used an in vivo model in which Rag2−/−γC−/− mice were repopulated with human hematopoietic stem cells. Using Rag2−/−γC−/− mice on a Nude background, we demonstrated that development and induction of SHMs of human IgM+IgD+CD27+ B cells can occur in a T cell–independent manner.

IL-7 Enhances Thymic Human T Cell Development in "Human Immune System" Rag2–/–IL-2Rγc–/– Mice without Affecting Peripheral T Cell Homeostasis

IL-7 is a central cytokine in the development of hematopoietic cells, although interspecies discrepancies have been reported. By coculturing human postnatal thymus hematopoietic progenitors and OP9-huDL1 stromal cells, we found that murine IL-7 is ∼100-fold less potent than human IL-7 for supporting human T cell development in vitro. We investigated the role of human IL-7 in newborn BALB/c Rag2−/−γc−/− mice transplanted with human hematopoietic stem cells (HSC) as an in vivo model of human hematopoiesis using three approaches to improve IL-7 signaling: administration of human IL-7, ectopic expression of human IL-7 by the transplanted human HSC, or enforced expression of a murine/human chimeric IL-7 receptor binding murine IL-7. We show that premature IL-7 signaling at the HSC stage, before entrance in the thymus, impeded T cell development, whereas increased intrathymic IL-7 signaling significantly enhanced the maintenance of immature thymocytes. Increased thymopoiesis was also observed when we transplanted BCL-2- or BCL-xL-transduced human HSC. Homeostasis of peripheral mature T cells in this humanized mouse model was not improved by any of these strategies. Overall, our results provide evidence for an important role of IL-7 in human T cell development in vivo and highlight the notion that IL-7 availability is but one of many signals that condition peripheral T cell homeostasis.

IL-6 triggers IL-21 production by human CD4+ T cells to drive STAT3-dependent plasma cell differentiation in B cells

Interleukin (IL)‐21‐producing CD4+T cells are central to humoral immunity. Deciphering the signals that induce IL‐21 production in CD4+ T cells and those triggered by IL‐21 in B cells are, therefore, of importance for understanding the generation of antibody (Ab) responses. Here, we show that IL‐6 increased IL‐21 production by human CD4+ T cells, particularly in those that express the transcriptional regulator B cell lymphoma (BCL)6, which is required in mice for the development of C‐X‐C chemokine receptor type 5 (CXCR5+) IL‐21‐producing T follicular helper (TFH) cells. However, retroviral overexpression of BCL6 in total human CD4+ T cells only transiently increased CXCR5, the canonical TFH‐defining surface marker. We show here that IL‐21 was required for the induction of Ab production by IL‐6. In IL‐21‐treated B cells, signal transducer and activator of transcription (STAT)3 was required for optimal immunoglobulin production and upregulation of PR domain containing 1 (PRDM1+), the master plasma cell factor. These results, therefore, demonstrate the critical importance of STAT3 activation in B cells during IL‐21‐driven humoral immunity and suggest that BCL6 expression, although not sufficient, may serve as a platform for the acquisition of a TFH‐like phenotype by human CD4+ T cells.

Spi-B inhibits human plasma cell differentiation by repressing BLIMP1 and XBP-1 expression.

The terminal differentiation of B cells into antibody-secreting plasma cells is tightly regulated by a complex network of transcription factors. Here we evaluated the role of the Ets factor Spi-B during terminal differentiation of human B cells. All mature tonsil and peripheral blood B-cell subsets expressed Spi-B, with the exception of plasma cells. Overexpression of Spi-B in CD19(+) B cells inhibited, similar to the known inhibitor BCL-6, the expression of plasma cell-associated surface markers and transcription factors as well as immunoglobulin production, ie, in vitro plasma cell differentiation. The arrest in B-cell differentiation enforced by Spi-B was independent of the transactivation domain, but dependent on the Ets-domain. By chromatin immunoprecipitation and assays using an inducible Spi-B construct BLIMP1 and XBP-1 were identified as direct target genes of Spi-B mediated repression. We propose a novel role for Spi-B in maintenance of germinal center and memory B cells by direct repression of major plasma cell factors and thereby plasma cell differentiation.

Functional Human Antigen-Specific T Cells Produced In Vitro Using Retroviral T Cell Receptor Transfer into Hematopoietic Progenitors

In vitro production of human T cells with known Ag specificity is of major clinical interest for immunotherapy against tumors and infections. We have performed TCRαβ gene transfer into human hemopoietic progenitors from postnatal thymus or umbilical cord blood, and subsequently cultured these precursors on OP9 stromal cells expressing the Notch human ligand Delta-like1. We report here that fully mature, functional T cells with controlled Ag specificity are obtained from such cultures. Using vectors encoding TCRαβ-chains directed against melanoma (MART-1), viral (CMV), and minor histocompatibility (HA-2) Ags, we show that the obtained Ag-specific T cells exert cytolytic activity against their cognate Ag and expand in vitro upon specific TCR stimulation. Therapeutic applications may arise from these results because they provide a way to produce large numbers of autologous mature Ag-specific T cells in vitro from undifferentiated hemopoietic progenitors.