Heike Schmidlin

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.

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.

New insights into the regulation of human B-cell differentiation

B lymphocytes provide the cellular basis of the humoral immune response. All stages of this process, from B-cell activation to formation of germinal centers and differentiation into memory B cells or plasma cells, are influenced by extrinsic signals and controlled by transcriptional regulation. Compared to naïve B cells, memory B cells display a distinct expression profile, which allows for their rapid secondary responses. Indisputably, many B-cell malignancies result from aberrations in the circuitry controlling B-cell function, particularly during the germinal centre (GC) reaction. Here, we review new insights into memory B-cell subtypes, recent literature on transcription factors regulating human B-cell differentiation and further evidence for B-cell lymphomagenesis emanating from errors during GC cell reactions.

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.

The transcription factor Spi-B regulates human plasmacytoid dendritic cell survival through direct induction of the antiapoptotic gene BCL2-A1

Plasmacytoid dendritic cells (pDCs) selectively express Toll-like receptor (TLR)-7 and TLR-9, which allow them to rapidly secrete massive amounts of type I interferons after sensing nucleic acids derived from viruses or bacteria. It is not completely understood how development and function of pDCs are controlled at the transcriptional level. One of the main factors driving pDC development is the ETS factor Spi-B, but little is known about its target genes. Here we demonstrate that Spi-B is crucial for the differentiation of hematopoietic progenitor cells into pDCs by controlling survival of pDCs and its progenitors. In search for Spi-B target genes, we identified the antiapoptotic gene Bcl2-A1 as a specific and direct target gene, thereby consolidating the critical role of Spi-B in cell survival.

Isolation and in vitro generation of gene-manipulated human plasmacytoid and conventional dendritic cells.

Our understanding of human lymphocyte development has increased significantly over the past 20 years. In particular, our insight into human T- and B-cell development has improved (1, 2). Nonetheless, there are many gaps in our understanding, particularly regarding the early stages of development of hematopoietic progenitor cells (HPCs) into downstream lineage-biased and lineage-restricted precursors and the molecular mechanisms underlying these activities. The same holds true for our knowledge of human dendritic cell (DC) development. While the amount of data on the different subsets of conventional DCs (cDCs) and plasmacytoid DCs (pDCs) rapidly increases in mice (3, 4), the developmental stages of different DC subsets in humans remain poorly defined (2). The relatively easy access to patient material and therefore human precursor cells that can be isolated from these tissues combined with the availability of in vitro and in vivo differentiation assays allows studies in the field of human hematopoietic development, including that of DCs. In addition, the opportunities to manipulate gene expression, by stable overexpression of a gene of interest or RNA interference-mediated knockdown, generate valuable information about the mechanisms underlying lineage commitment and differentiation.