All Publications

  • IL-6 triggers IL-21 production by human CD4+ T cells to drive STAT3-dependent plasma cell differentiation in B cells. Immunology and cell biology Diehl, S. A., Schmidlin, H., Nagasawa, M., Blom, B., Spits, H. 2012; 90 (8): 802-11


    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 (T(FH)) cells. However, retroviral overexpression of BCL6 in total human CD4(+) T cells only transiently increased CXCR5, the canonical T(FH)-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 T(FH)-like phenotype by human CD4(+) T cells.

    View details for DOI 10.1038/icb.2012.17

    View details for PubMedID 22491065

    View details for PubMedCentralID PMC3396759

  • The transcription factor Spi-B regulates human plasmacytoid dendritic cell survival through direct induction of the antiapoptotic gene BCL2-A1. Blood Karrich, J. J., Balzarolo, M., Schmidlin, H., Libouban, M., Nagasawa, M., Gentek, R., Kamihira, S., Maeda, T., Amsen, D., Wolkers, M. C., Blom, B. 2012; 119 (22): 5191-200


    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.

    View details for DOI 10.1182/blood-2011-07-370239

    View details for PubMedID 22510878

  • Isolation and in vitro generation of gene-manipulated human plasmacytoid and conventional dendritic cells. Methods in molecular biology (Clifton, N.J.) Schotte, R., Schmidlin, H., Nagasawa, M., Dontje, W., Karrich, J. J., Uittenbogaart, C., Spits, H., Blom, B. 2010; 595: 67-85


    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.

    View details for DOI 10.1007/978-1-60761-421-0_5

    View details for PubMedID 19941106

    View details for PubMedCentralID PMC4266553

  • New insights into the regulation of human B-cell differentiation. Trends in immunology Schmidlin, H., Diehl, S. A., Blom, B. 2009; 30 (6): 277-85


    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.

    View details for DOI 10.1016/

    View details for PubMedID 19447676

    View details for PubMedCentralID PMC2792751

  • Spi-B inhibits human plasma cell differentiation by repressing BLIMP1 and XBP-1 expression BLOOD Schmidlin, H., Diehl, S. A., Nagasawa, M., Scheeren, F. A., Schotte, R., Uittenbogaart, C. H., Spits, H., Blom, B. 2008; 112 (5): 1804-1812


    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.

    View details for DOI 10.1182/blood-2008-01-136440

    View details for Web of Science ID 000258956200037

    View details for PubMedID 18552212

    View details for PubMedCentralID PMC2518887

  • 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. European journal of immunology Nagasawa, M., Schmidlin, H., Hazekamp, M. G., Schotte, R., Blom, B. 2008; 38 (9): 2389-400


    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.

    View details for DOI 10.1002/eji.200838470

    View details for PubMedID 18792017

  • STAT3-mediated up-regulation of BLIMP1 is coordinated with BCL6 down-regulation to control human plasma cell differentiation JOURNAL OF IMMUNOLOGY Diehl, S. A., Schmidlin, H., Nagasawa, M., van Haren, S. D., Kwakkenbos, M. J., Yasuda, E., Beaumont, T., Scheeren, F. A., Spits, H. 2008; 180 (7): 4805-4815


    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.

    View details for Web of Science ID 000257506700053

    View details for PubMedID 18354204

    View details for PubMedCentralID PMC2396731

  • Stimulated plasmacytoid dendritic cells impair human T-cell development. Blood Schmidlin, H., Dontje, W., Groot, F., Ligthart, S. J., Colantonio, A. D., Oud, M. E., Schilder-Tol, E. J., Spaargaren, M., Spits, H., Uittenbogaart, C. H., Blom, B. 2006; 108 (12): 3792-800


    Thymic plasmacytoid dendritic cells (pDCs) are located predominantly in the medulla and at the corticomedullary junction, the entry site of bone marrow-derived multipotential precursor cells into the thymus, allowing for interactions between thymic pDCs and precursor cells. We demonstrate that in vitro-generated pDCs stimulated with CpG or virus impaired the development of human autologous CD34(+)CD1a(-) thymic progenitor cells into the T-cell lineage. Rescue by addition of neutralizing type I interferon (IFN) antibodies strongly implies that endogenously produced IFN-alpha/beta is responsible for this inhibitory effect. Consistent with this notion, we show that exogenously added IFN-alpha had a similar impact on IL-7- and Notch ligand-induced development of thymic CD34(+)CD1a(-) progenitor cells into T cells, because induction of CD1a, CD4, CD8, and TCR/CD3 surface expression and rearrangements of TCRbeta V-DJ gene segments were severely impaired. In addition, IL-7-induced proliferation but not survival of the developing thymic progenitor cells was strongly inhibited by IFN-alpha. It is evident from our data that IFN-alpha inhibits the IL-7R signal transduction pathway, although this could not be attributed to interference with either IL-7R proximal (STAT5, Akt/PKB, Erk1/2) or distal (p27(kip1), pRb) events.

    View details for DOI 10.1182/blood-2006-02-004978

    View details for PubMedID 16917011

    View details for PubMedCentralID PMC1895464

  • On the relevance of TCR rearrangement circles as molecular markers for thymic output during experimental graft-versus-host disease. Journal of immunology (Baltimore, Md. : 1950) Krenger, W., Schmidlin, H., Cavadini, G., Holländer, G. A. 2004; 172 (12): 7359-67


    Efficient reconstitution of the pool of peripheral T cells after hemopoietic stem cell transplantation (HSCT) is dependent on normal thymic function. However, the development of graft-vs-host disease (GVHD) in the context of allogeneic HSCT is associated with injurious effects on thymocyte development. In this study, we examined in models of syngeneic and allogeneic murine HSCT whether actual posttransplant thymic output is accurately reflected by analysis of signal-joint TCR rearrangement excision circles (sjTRECs). Our data demonstrate that the de novo generation of T cells following syngeneic HSCT of T cell-deficient B6.RAG2(-/-) (recombination-activating gene 2(-/-)) mice correlates firmly with an increase of sjTRECs in the thymus and spleen. However, the altered homeostasis of naive peripheral T cells in the presence of GVHD necessitates the combined analysis of cell division in vivo and determinations of sjTREC contents and total sjTREC numbers to draw informative conclusions. From our data, we substantiate that thymic output and peripheral division of newly generated T cells are diminished in the presence of acute GVHD in an experimental radiation/allogeneic HSCT model.

    View details for DOI 10.4049/jimmunol.172.12.7359

    View details for PubMedID 15187112