APRIL secreted by neutrophils binds to heparan sulfate proteoglycans to create plasma cell niches in human mucosa.

The bone marrow constitutes a favorable environment for long-lived antibody-secreting plasma cells, providing blood-circulating antibody. Plasma cells are also present in mucosa-associated lymphoid tissue (MALT) to mediate local frontline immunity, but how plasma cell survival there is regulated is not known. Here we report that a proliferation-inducing ligand (APRIL) promoted survival of human upper and lower MALT plasma cells by upregulating expression of the antiapoptotic proteins bcl-2, bcl-xL, and mcl-1. The in situ localization of APRIL was consistent with such a prosurvival role in MALT. In upper MALT, tonsillar epithelium produced APRIL. Upon infection, APRIL production increased considerably when APRIL-secreting neutrophils recruited from the blood infiltrated the crypt epithelium. Heparan sulfate proteoglycans (HSPGs) retained secreted APRIL in the subepithelium of the infected zone to create APRIL-rich niches, wherein IgG-producing plasma cells accumulated. In lower MALT, neutrophils were the unique source of APRIL, giving rise to similar niches for IgA-producing plasmocytes in villi of lamina propria. Furthermore, we found that mucosal humoral immunity in APRIL-deficient mice is less persistent than in WT mice. Hence, production of APRIL by inflammation-recruited neutrophils may create plasma cell niches in MALT to sustain a local antibody production.

Ex vivo expansion of haematopoietic stem cells and gene therapy development.

The results of a research project on ex vivo expansion of human haematopoietic stem cells (HSC) and development of gene therapy, funded by the Swiss National Research Program 46, are summarised and discussed in the context of current progresses and difficulties in these fields. A routine method for ex vivo expansion of human HSC is not yet available. However, stem cell biology has progressed importantly in recent years; ex vivo expansion of human HSC should become possible in the near future. Regarding gene therapy development, we obtained with HIV-1-derived bicistronic lentiviral vectors efficient delivery of genes into immature haematopoietic cells and also primary human B lymphocytes. However, clinical gene therapy still faces a variety of problems. For the (into chromosomes) integrating lentivectors, currently the most promising tools for HSC-based gene therapy, the risks of insertional mutagenesis need to be fully assessed before larger clinical trials can start.

Ex vivo expansion of hematopoietic stem cells and gene therapy development.

The results of a research project on ex vivo expansion of human haematopoietic stem cells (HSC) and development of gene therapy, funded by the Swiss National Research Program 46, are summarised and discussed in the context of current progresses and difficulties in these fields. A routine method for ex vivo expansion of human HSC is not yet available. However, stem cell biology has progressed importantly in recent years; ex vivo expansion of human HSC should become possible in the near future. Regarding gene therapy development, we obtained with HIV-1-derived bicistronic lentiviral vectors efficient delivery of genes into immature haematopoietic cells and also primary human B lymphocytes. However, clinical gene therapy still faces a variety of problems. For the (into chromosomes) integrating lentivectors, currently the most promising tools for HSC-based gene therapy, the risks of insertional mutagenesis need to be fully assessed before larger clinical trials can start.

Transduction of CpG DNA-stimulated primary human B cells with bicistronic lentivectors.

Recently, using HIV-1-derived lentivectors, we obtained efficient transduction of primary human B lymphocytes cocultured with murine EL-4 B5 thymoma cells, but not of isolated B cells activated by CD40 ligation. Coculture with a cell line is problematic for gene therapy applications or study of gene functions. We have now found that transduction of B cells in a system using CpG DNA was comparable to that in the EL-4 B5 system. A monocistronic vector with a CMV promoter gave 32 +/- 4.7% green fluorescent protein (GFP)+ cells. A bicistronic vector, encoding IL-4 and GFP in the first and second cistrons, respectively, gave 14.2 +/- 2.1% GFP+ cells and IL-4 secretion of 1.3 +/- 0.2 ng/10(5) B cells/24 h. This was similar to results obtained in CD34+ cells using the elongation factor-1alpha promoter. Activated memory and naive B cells were transducible. After transduction with a bicistronic vector encoding a viral FLIP molecule, vFLIP was detectable by FACS or Western blot in GFP+, but not in GFP-, B cells, and 57% of sorted GFP+ B cells were protected against Fas ligand-induced cell death. This system should be useful for gene function research in primary B cells and development of gene therapies.

TNF-alpha induces the generation of Langerin/(CD207)+ immature Langerhans-type dendritic cells from both CD14-CD1a and CD14+CD1a- precursors derived from CD34+ cord blood cells.

CD34+ cell-derived hematopoietic precursors amplified with FLT3-ligand, thrombopoietin and stem cell factor became, after a 6-day induction with GM-CSF, IL-4 and TGF-beta1, HLA-DR+, CD1a+, CD83-, CD86-, CD80- cells. A fraction of them expressed Langerin, Lag, and E-cadherin, resembling epidermal Langerhans cells (LC). TNF-alpha added for the last 3 days only marginally induced CD83 expression, but strikingly increased the proportion of immature Langerin+CD83- LC. Langerin+CD83+ and Langerin+CD83- cells were functionally distinct, the former internalizing less efficiently Langerin than the latter. Both CD1a-CD14- and CD1a-CD14+ cells sorted from FLT3-ligand, thrombopoietin and stem cell factor cultures responded to TNF-alpha by an increase of Langerin+ cells. Thus, TNF-alpha rescued LC precursors irrespective of their commitment to the monocytic lineage. When added to GM-CSF, IL-4 and TGF-beta1 containing-cultures, LPS or IL-1beta also induced significant numbers of Langerin+CD83- immature cells displaying a low allostimulatory activity, while CD40-ligand largely promoted highly allostimulatory Langerin-CD83+ cells. Altogether, these data show that in contrast to CD40-ligand, which induced LC maturation even in presence of TGF-beta1, nonspecific proinflammatory factors such as TNF-alpha, IL-1 or LPS, essentially induced immature LC generation, and little cell activation in the presence of TGF-beta1.

Efficient transduction of primary human B lymphocytes and nondividing myeloma B cells with HIV-1-derived lentiviral vectors.

We studied the transduction of primary human B lymphocytes and myeloma cells with lentiviral vectors. In peripheral blood B cells that had been activated with helper T cells (murine thymoma EL-4 B5) and cytokines, multiply attenuated HIV-1-derived vectors pseudotyped with vesicular stomatitis virus (VSV) G-envelope protein achieved the expression of green fluorescence protein (GFP) in 27% +/- 12% (mean +/- 1 SD; median, 27%) of B cells in different experiments. When compared in parallel cultures, the transducibility of B cells from different donors exhibited little variation. The human cytomegalovirus (CMV) promoter gave 4- to 6-fold higher GFP expression than did the human elongation factor-1alpha promoter. A murine retroviral vector pseudotyped with VSV G protein proved inefficient even in mitotically active primary B cells. B cells freshly stimulated with Epstein-Barr virus were also transducible by HIV vectors (24% +/- 9%), but B cells activated with CD40 ligand and cytokines resisted transduction. Thus, different culture systems gave different results. Freshly isolated, nondividing myeloma cells were efficiently transduced by HIV vectors; for 6 myelomas the range was 14% to 77% (median, 28%) GFP(+) cells. HIV vectors with a mutant integrase led to no significant GFP signal in primary B or myeloma cells, suggesting that vector integration was required for high transduction. In conclusion, HIV vectors are promising tools for studies of gene functions in primary human B cells and myeloma cells for the purposes of research and the development of gene therapies.