Bianca Blom

Novel p19 Protein Engages IL-12p40 to Form a Cytokine, IL-23, with Biological Activities Similar as Well as Distinct from IL-12

A novel sequence discovered in a computational screen appears distantly related to the p35 subunit of IL-12. This factor, which we term p19, shows no biological activity by itself; instead, it combines with the p40 subunit of IL-12 to form a novel, biologically active, composite cytokine, which we term IL-23. Activated dendritic cells secrete detectable levels of this complex. IL-23 binds to IL-12R beta 1 but fails to engage IL-12R beta 2; nonetheless, IL-23 activates Stat4 in PHA blast T cells. IL-23 induces strong proliferation of mouse memory (CD4(+)CD45Rb(low)) T cells, a unique activity of IL-23 as IL-12 has no effect on this cell population. Similar to IL-12, human IL-23 stimulates IFN-gamma production and proliferation in PHA blast T cells, as well as in CD45RO (memory) T cells.

The Transcription Factor GATA3 Is Essential for the Function of Human Type 2 Innate Lymphoid Cells

Type 2 innate lymphoid cells (ILC2s) are part of a large family of ILCs that are important effectors in innate immunity, lymphoid organogenesis, and tissue remodeling. ILC2s mediate parasite expulsion but also contribute to airway inflammation, emphasizing the functional similarity between these cells and Th2 cells. Consistent with this, we report that the transcription factor GATA3 was highly expressed by human ILC2s. CRTH2(+) ILC2s were enriched in nasal polyps of patients with chronic rhinosinusitis, a typical type 2-mediated disease. Nasal polyp epithelial cells expressed TSLP, which enhanced STAT5 activation, GATA3 expression, and type 2 cytokine production in ILC2s. Ectopic expression of GATA3 in Lin(-)CD127(+)CRTH2(-) cells resulted in induction of CRTH2 and the capacity to produce high amounts of type 2 cytokines in response to TSLP plus IL-33. Hence, we identify GATA3, potently regulated by TSLP, as an essential transcription factor for the function of human ILC2s.

DAP12-Deficient Mice Fail to Develop Autoimmunity Due to Impaired Antigen Priming

DAP12 is an ITAM-bearing membrane adaptor molecule implicated in the activation of NK and myeloid cells. In mice rendered DAP12 deficient by targeted gene disruption, lymphoid and myeloid development was apparently normal, although the activating Ly49 receptors on NK cells were downregulated and nonfunctional. To analyze the consequences of DAP12 deficiency in vivo, we examined the susceptibility of DAP12-/- mice to experimental autoimmune encephalomyelitis (EAE). DAP12-/- mice were resistant to EAE induced by immunization with myelin oligodendrocyte glycoprotein (MOG) peptide. Resistance was associated with a strongly diminished production of IFNgamma by myelin-reactive CD4+ T cells due to inadequate T cell priming in vivo. These data suggest that DAP12 signaling may be required for optimal antigen-presenting cell (APC) function or inflammation.

Interleukin-12 and -23 Control Plasticity of CD127+ Group 1 and Group 3 Innate Lymphoid Cells in the Intestinal Lamina Propria

Human group 1 ILCs consist of at least three phenotypically distinct subsets, including NK cells, CD127(+) ILC1, and intraepithelial CD103(+) ILC1. In inflamed intestinal tissues from Crohns disease patients, numbers of CD127(+) ILC1 increased at the cost of ILC3. Here we found that differentiation of ILC3 to CD127(+) ILC1 is reversible in vitro and in vivo. CD127(+) ILC1 differentiated to ILC3 in the presence of interleukin-2 (IL-2), IL-23, and IL-1β dependent on the transcription factor RORγt, and this process was enhanced in the presence of retinoic acid. Furthermore, we observed in resection specimen from Crohns disease patients a higher proportion of CD14(+) dendritic cells (DC), which in vitro promoted polarization from ILC3 to CD127(+) ILC1. In contrast, CD14(-) DCs promoted differentiation from CD127(+) ILC1 toward ILC3. These observations suggest that environmental cues determine the composition, function, and phenotype of CD127(+) ILC1 and ILC3 in the gut.

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.

Thrombopoietin cooperates with FLT3-ligand in the generation of plasmacytoid dendritic cell precursors from human hematopoietic progenitors

Type 1 interferon-producing cells (IPCs), also known as plasmacytoid dendritic cell (DC) precursors, represent the key effectors in antiviral innate immunity and triggers for adaptive immune responses. IPCs play important roles in the pathogenesis of systemic lupus erythematosus (SLE) and in modulating immune responses after hematopoietic stem cell transplantation. Understanding IPC development from hematopoietic progenitor cells (HPCs) may provide critical information in controlling viral infection, autoimmune SLE, and graft-versus-host disease. FLT3-ligand (FLT3-L) represents a key IPC differentiation factor from HPCs. Although hematopoietic cytokines such as interleukin-3 (IL-3), IL-7, stem cell factor (SCF), macrophage-colony-stimulating factor (M-CSF), and granulocyte M-CSF (GM-CSF) promote the expansion of CD34+ HPCs in FLT3-L culture, they strongly inhibit HPC differentiation into IPCs. Here we show that thrombopoietin (TPO) cooperates with FLT3-L, inducing CD34+ HPCs to undergo a 400-fold expansion in cell numbers and to generate more than 6 x 10(6) IPCs per 10(6) CD34+ HPCs within 30 days in culture. IPCs derived from HPCs in FLT3-L/TPO cultures display blood IPC phenotype and have the capacity to produce large amounts of interferon-alpha (IFN-alpha) and to differentiate into mature DCs. This culture system, combined with the use of adult peripheral blood CD34+ HPCs purified from G-CSF-mobilized donors, permits the generation of more than 10(9) IPCs from a single blood donor.

IL-21 is expressed in Hodgkin lymphoma and activates STAT5: evidence that activated STAT5 is required for Hodgkin lymphomagenesis

Classical Hodgkin lymphoma (HL) is a malignant disorder characterized by the presence of neoplastic mononucleated Hodgkin and multinucleated Reed-Sternberg cells. Here, we show that both the interleukin (IL)-21 receptor as well as IL-21 are expressed by HL cells. IL-21 activates signal transducer of activation and transcription 3 (STAT3) and STAT5 in HL cell lines and activated human B cells. Ectopic expression of constitutively active STAT5 in primary human B cells resulted in immortalized B cells that have lost the B-cell phenotype and strongly resembled HL cells, which could partially be rescued by ectopic expression of the B cell-determining transcription factor E47. Data from experiments using reporter assays and overexpression of constitutively active IKK2 support the hypothesis that the STAT5 and nuclear factor-kappaB (NF-kappaB) pathways collaborate in HL genesis.

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.

Endogenous IFN-α Production by Plasmacytoid Dendritic Cells Exerts an Antiviral Effect on Thymic HIV-1 Infection

Plasmacytoid dendritic cells (pDC) are the principal producers of IFN-α in response to viral infection. Because pDC are present in the thymus, we investigated the consequences of HIV-1-induced IFN-α production by thymic pDC. We observed that thymic pDC as well as thymocytes express intracellular IFN-α upon infection with HIV-1. However, only the pDC could suppress HIV-1 replication, because depletion of pDC resulted in enhancement of HIV-1 replication in thymocytes. Thymic pDC could also produce IFN-α in response to CpG oligonucleotides, consistent with the observations of others that peripheral pDC produce IFN-α upon engagement of TLR-9. Importantly, CpG considerably increased IFN-α production induced by HIV-1, and addition of CpG during HIV-1 infection enhanced expression of the IFN response protein MxA in thymocytes and strongly reduced HIV-replication. Our data indicate that thymic pDC modulate HIV-1 replication through secretion of IFN-α. The degree of inhibition depends on the level of IFN-α produced by the thymic pDC.

Evaluation of safety and efficacy of RNAi against HIV-1 in the human immune system (Rag-2(-/-)gammac(-/-)) mouse model

RNA interference (RNAi) gene therapy against HIV-1 by stable expression of antiviral short hairpin RNAs (shRNAs) can potently inhibit viral replication in T cells. Recently, a mouse model with a human immune system (HIS) was developed that can be productively infected with HIV-1. In this in vivo model, in which Rag-2−/−γc−/− mice are engrafted with human CD34+CD38− hematopoietic precursor cells, we evaluated an anti-HIV RNAi gene therapy. Human hematopoietic stem cells were transduced with a lentiviral vector expressing an shRNA against the HIV-1 nef gene (shNef) or the control vector. We observed normal development of the different cell subsets of the immune system. However, although initial transduction efficiencies were similar for both vectors, a reduced percentage of transduced human immune cells was observed for the shNef vector after establishment of the HIS in vivo. Further studies are required to fully evaluate the safety implications. When we infected the mature human CD4+ T cells from the HIS mouse ex vivo with HIV-1, potent inhibition of viral replication was scored in shNef-expressing cells, confirming efficacy. When challenged with an shNef-resistant HIV-1 variant, equal replication was scored in control and shNef-expressing cells, confirming sequence-specificity of the RNAi therapy. We thus demonstrated that an antiviral RNAi-based gene therapy on blood stem cells leads to HIV-1-resistant T cells in vivo, an important proof of concept in the clinical development of RNAi against HIV-1.